Modulation of CD4+ T cell responses by a tick saliva protein, Salp15 and polypeptides derived therefrom

ABSTRACT

Salp15, biologically functional equivalents and fragments thereof, and nucleic acid molecules encoding the same are disclosed. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also disclosed. Salp15 gene products and Salp15 polypeptide fragments have biological activity in modulating CD4+ T cell activation through specific binding to CD4. Thus, therapeutic methods involving modulating T cell activation using Salp15 and biologically active polypeptide fragments thereof are also disclosed. The specific binding of Salp15 and fragment peptides thereof to CD4 can inhibit HIV infection of T cells, and thus methods of using Salp15 for inhibiting HIV infection are also disclosed. Screening methods for selecting substances having an ability to modulate activation of T cells are also disclosed.

RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.11/667,748, filed Aug. 1, 2008; which claimed the benefit of PCTInternational Patent Application Serial No. PCT/US2005/032843, filedSep. 14, 2005; which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/627,122, filed Nov. 12, 2004. The disclosure ofeach of these applications is incorporated herein by reference in itsentirety.

GOVERNMENT INTEREST

This invention was made with U.S. Government support under Grant No.R01AI053064 awarded by National Institute of Allergy and InfectiousDiseases, National Institutes of Health. The U.S. Government has certainrights in the presently disclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter relates generally to isolated andpurified polypeptides and nucleic acids and methods of using same. Moreparticularly, the presently disclosed subject matter relates to isolatedand purified Salp15 and biologically active fragments thereof havingbinding specificity for CD4 receptor polypeptides, and purified nucleicacid molecules encoding same. The presently disclosed subject matterfurther relates to methods of using the polypeptides to modulateactivation of CD4⁺ T cells, including therapeutic methods for treatingdisorders related to T cell activation as well as inhibiting T cellinfection by HIV. The presently disclosed subject matter further relatesto screening methods for selecting compositions that can modulateactivation of CD4⁺ T cells.

BACKGROUND

The immune system is highly complex and tightly regulated, with manyalternative pathways capable of compensating for deficiencies in otherparts of the system. There are however occasions when the immuneresponse becomes a cause of disease or other undesirable conditions ifactivated. Immunoinflammatory disorders are thus characterized generallyby the inappropriate activation of the body's immune defenses. Ratherthan targeting infectious invaders, the immune response targets anddamages the body's own tissues or transplanted tissues. Such diseases orundesirable conditions are for example autoimmune diseases, graftrejection after transplantation, or allergy to innocuous antigens,psoriasis, chronic inflammatory diseases such as atherosclerosis, andinflammation in general.

The tissue targeted by the immune system varies with the disorder. Forexample, in multiple sclerosis, the immune response is directed againstthe neuronal tissue, while in Crohn's disease the digestive tract istargeted. Immunoinflammatory disorders affect millions of individualsand include conditions such as asthma, allergic intraocular inflammatorydiseases, rheumatoid arthritis, atopic dermatitis, atopic eczema, type Idiabetes, hemolytic anemia, inflammatory dermatoses, inflammatory bowelor gastrointestinal disorders (e.g., Crohn's disease and ulcerativecolitis), multiple sclerosis, myasthenia gravis, pruritis/inflammation,psoriasis, cirrhosis, and systemic lupus erythematosus.

In these cases and others involving inappropriate or undesired immuneresponse there is a clinical need for immunosuppression. The pathwaysleading to these undesired immune responses are numerous and in manycases are not fully elucidated. However, they often involve a commonstep, which is activation of lymphocytes.

Current treatment regimens for immunoinflammatory disorders typicallyrely on immunosuppressive agents that often are non-specific in theiractivity. The effectiveness of these agents can vary and their use isoften accompanied by adverse side effects. Thus, improved therapeuticagents having specificity for inhibiting activation of T lymphocytes andmethods for the treatment of immunoinflammatory disorders are needed.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In one embodiment, the presently disclosed subject matter provides anisolated and purified biologically active Salp15 polypeptide, comprising(a) a polypeptide encoded by a nucleic acid sequence as set forth in anyof SEQ ID NOs: 16 and 17, (b) a polypeptide encoded by a nucleic acidhaving at least about 90% or greater sequence identity to a DNA sequenceas set forth in any of SEQ ID NOs: 16 and 17, (c) a polypeptide havingan amino acid sequence of any of SEQ ID NOs: 13 and 14, or abiologically functional equivalent thereof, (d) a polypeptide which isimmunologically cross-reactive with antibodies which are immunologicallyreactive with a polypeptide having an amino acid sequence of any of SEQID NOs: 13 and 14, or (e) a polypeptide comprising a fragment of apolypeptide of (a), (b), (c), or (d). In some embodiments, the Salp15polypeptide is modified to be in detectably labeled form. In someembodiments, a composition comprising the Salp15 polypeptide and acarrier is provided. In some embodiments, the carrier is apharmaceutically acceptable carrier in humans.

In another embodiment of the presently disclosed subject matter, anisolated nucleic acid molecule is provided, comprising (a) a nucleicacid molecule encoding a polypeptide of any of SEQ ID NOs: 13 and 14,(b) a nucleic acid molecule having at least about 90% or greatersequence identity to a nucleic acid sequence as set forth in any of SEQID NOs: 16 and 17, or (c) a nucleic acid molecule having a sequence asset forth in any of SEQ ID NOs: 16 and 17. In some embodiments, arecombinant vector comprising the isolated nucleic acid moleculeoperatively linked to a promoter is provided, and in some embodiments arecombinant host cell comprising the nucleic acid molecule is furtherprovided.

In yet another embodiment, the presently disclosed subject matterprovides a method of modulating activation of a CD4⁺ T cell due to Tcell receptor-mediated signaling. In some embodiments, the methodcomprises contacting the T cell with a Salp15 polypeptide disclosedherein, where activation of the T cell is modulated. In someembodiments, modulating activation of the T cell comprises inhibitingactivation of the T cell, and in some embodiments inhibiting activationof the T cell results in decreased proliferation of the T cell. In someembodiments, contacting the T cell with the polypeptide comprisescontacting a CD4 receptor expressed on the surface of the T cell withthe polypeptide. Further, in some embodiments, contacting the CD4receptor with the polypeptide comprises contacting at least a region ofthe extracellular outer two domains (D1-D2) of the CD4 receptor with thepolypeptide. In some embodiments, the T cell is within a subject and thepolypeptide is administered to the subject. Further, in someembodiments, the polypeptide is administered by systemic administration,parenteral administration, oral delivery, buccal delivery, subcutaneousadministration, inhalation, intratracheal installation, surgicalimplantation, transdermal delivery, local injection, hyper-velocityinjection/bombardment, or combinations thereof.

In a still further embodiment, a method of treating a subject sufferingfrom or at risk of suffering from a condition characterized by a CD4⁺ Tcell response is provided. In some embodiments, the method comprisesadministering to the subject an effective amount of a Salp15 polypeptidedisclosed herein. In some embodiments, the condition is an autoimmunedisorder or a tissue or organ transplant rejection. Further, in someembodiments, the condition is an autoimmune disorder selected from thegroup consisting of lupus, rheumatoid arthritis, type 1 diabetes,multiple sclerosis, rheumatic fever, and Hashimoto's disease. In someembodiments, the polypeptide is administered by systemic administration,parenteral administration, oral delivery, buccal delivery, subcutaneousadministration, inhalation, intratracheal installation, surgicalimplantation, transdermal delivery, local injection, hyper-velocityinjection/bombardment, or combinations thereof.

In yet another embodiment of the presently disclosed subject matter, amethod of treating multiple sclerosis in a subject is provided. In someembodiments, the method comprises administering to the subject aneffective amount of a Salp15 polypeptide disclosed herein, or abiologically active fragment thereof, having immunosuppressive activityto the subject. In some embodiments, the Salp15 polypeptide isadministered by systemic administration, parenteral administration, oraldelivery, buccal delivery, subcutaneous administration, inhalation,intratracheal installation, surgical implantation, transdermal delivery,local injection, hyper-velocity injection/bombardment, or combinationsthereof. In some embodiments, treating the multiple sclerosis comprisestreating a relapsing episode of multiple sclerosis resulting fromepitope spreading.

In yet another embodiment, the presently disclosed subject matterprovides a method of inhibiting infection of a T cell by a humanimmunodeficiency virus (HIV). In some embodiments, the method comprisescontacting a CD4 receptor expressed by the T cell with a Salp15polypeptide disclosed herein, or a biologically active fragment thereofhaving binding specificity for the CD4 receptor, whereby contacting theSalp15 polypeptide with the CD4 receptor inhibits the HIV from infectingthe T cell. In some embodiments, contacting the CD4 receptor with theSalp15 polypeptide comprises contacting the extracellular outer twodomains (D1-D2) of the CD4 receptor with the Salp15 polypeptide. In someembodiments, the T cell is within a subject and the Salp15 polypeptideis administered to the subject. Further, in some embodiments, thepolypeptide is administered by systemic administration, parenteraladministration, oral delivery, buccal delivery, subcutaneousadministration, inhalation, intratracheal installation, surgicalimplantation, transdermal delivery, local injection, hyper-velocityinjection/bombardment, or combinations thereof.

In another embodiment, a method of screening a candidate substance foran ability to modulate activation of a CD4⁺ T cell due to T cellreceptor-mediated signaling is provided. In some embodiments, the methodcomprises establishing a test sample comprising a CD4 receptorpolypeptide and a ligand for the CD4 receptor polypeptide, wherein theligand is a Salp15 polypeptide disclosed herein; administering acandidate substance to the test sample; and measuring the effect of thecandidate substance on binding of the CD4 receptor polypeptide and theligand in the test sample to thereby determine the ability of thecandidate substance to modulate activation of a CD4⁺ T cell due to Tcell receptor-mediated signaling. In some embodiments, the test samplefurther comprises an indicator, and the ability of the candidatesubstance to modulate activation of a CD4⁺ T cell is determined bydetecting a signal produced by the indicator upon an effect of thecandidate substance on binding of the CD4 receptor polypeptide and theligand; and identifying the candidate substance as a modulator ofactivation of a CD4⁺ T cell based upon an amount of signal produced ascompared to a control sample. In some embodiments, the candidatesubstance is a candidate polypeptide. In some embodiments, the ligandcomprises an indicator. In some embodiments, the method furthercomprises the step of purifying and isolating a nucleic acid moleculeencoding the candidate polypeptide. In some embodiments, the candidatepolypeptide is an antibody or biologically functional equivalentfragment thereof and in other embodiments, the candidate substance is asmall molecule. In some embodiments, the CD4 receptor polypeptide isimmobilized to a solid support.

Therefore, it is an object of the presently disclosed subject matter toprovide compositions comprising a Salp15 polypeptide which can modulateCD4⁺ T cell responses.

An object of the presently disclosed subject matter having been statedhereinabove, and which is addressed in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingdrawings and EXAMPLES as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show Salp15 inhibits protein tyrosine phosphorylation duringT cell activation.

FIG. 1A shows PLCγ1 phosphorylation is decreased in anti-CD3/CD28induced mouse CD4⁺ T cells in the presence of Salp15, as analyzed byimmunoblotting (upper panel) and confocal microscopy (lower panel) usinganti-pPLCγ1⁷⁸³.

FIG. 1B is a panel of representative immunofluorescence micrographsshowing staining with anti-pTyr (pY) and anti-pLat¹⁹¹ (pLAT) in CD4⁺ Tcells stimulated in the presence or absence of Salp15.

FIG. 1C shows a set of Western blots showing the decrease in tyrosinephosphorylation of Lck, Zap70 and Vav1 in CD4⁺ T cells stimulated in thepresence of Salp15.

FIGS. 2A-2C show Salp15 inhibits lipid raft redistribution in activatedCD4⁺ T cells.

FIG. 2A shows photomicrographs of CTB594 staining in CD4⁺ T cellsstimulated in the presence or absence of Salp15.

FIG. 2B shows photomicrographs of Salp15 treatment affects receptorcapping in CD4⁺ T cells but not in macrophages and B cells afteractivation using anti-CD3/CD28 cross-linked with anti-hamster IgG₄₈₈,anti-CD16/CD32 cross-linked with anti-rat IgG₅₉₄ and anti-IgMcross-linked with anti-rat IgG₅₉₄, respectively.

FIG. 2C shows photomicrographs of CTB₅₉₄ staining in CD8⁺ T cellsstimulated with anti-CD3/CD28 in the presence or absence of Salp15.

FIGS. 3A-3C show Salp15 pretreatment reduces actin polymerization duringT cell activation.

FIG. 3A shows photomicrographs of Phalloidin₄₈₈ staining in CD4⁺ T cellsstimulated in the presence or absence of Salp15.

FIG. 3B shows that the levels of F-actin and G-actin isolated fromlysates of CD4⁺ T cells treated as indicated, were determined byimmunoblotting with anti-actin. The lower panel shows the respectivelevels of total actin in the cell lysates.

FIG. 3C is a graph showing flow cytometric analysis of F-actin formationin unstimulated (shaded area) or stimulated CD4⁺ T cells in the presence(gray) or absence (black) of Salp15 stained with phalloidin₄₈₈.

FIGS. 4A-4G show Salp15 binds directly to CD4.

FIG. 4A shows Jurkat cell lysate containing His-tagged Salp15 wasimmunoprecipitated using anti-His antibodies or IgG (control). Theimmunoprecipitate was subjected to Western blotting using anti-His andanti-CD4 antibodies.

FIG. 4B shows confocal photomicrographs demonstrating co-localization ofanti-CD4 staining and Salp15₄₈₈ binding on naïve and stimulated CD4⁺ Tcells.

FIG. 4C shows graphs depicting flow cytometric analysis of CD4expression (left panel) and Salp15₄₈₈ binding (right panel) in HeLa(black shaded) and HeLa-CD4 (unshaded) cells.

FIG. 4D shows co-localization of CD4 and Salp15₄₈₈ binding on HeLa-CD4cells in photomicrographs.

FIG. 4E shows results of HeLa and HeLa-CD4 cell lysates containingSalp15 after immunoprecipitation using anti-His Ab. Theimmunoprecipitates and aliquots of both HeLa and HeLa-CD4 cell lysates(WCE) were subjected to immunoblotting using anti-CD4 and anti-Hisantibodies. Reciprocal immunoprecipitation from HeLa-CD4 cell lysate wasdone using anti-CD4 or IgG followed by immunoblotting with anti-CD4 oranti-His antibodies.

FIG. 4F shows photomicrographs demonstrating that unlabeled Salp15 butnot lysozyme pretreatment of HeLa-CD4 cells blocks Salp15₄₈₈ binding.

FIG. 4G shows photomicrographs demonstrating that preincubation ofHeLa-CD4 cells with polyclonal anti-CD4 abolishes Salp15₄₈₈ binding ascompared to monoclonal OKT4. MT310 mAb also exhibited competition withSalp15₄₈₈ binding on HeLa-CD4 cells.

FIGS. 5A-5C show Salp15 binds to the outer-most extracellular domains ofCD4.

FIG. 5A is a graph of Salp15 (0.4:M) incubated with increasing amountsof immobilized sCD4 (extracellular domains D1D2, squares) or lysozyme(triangles) in a microtiter assay showing saturable binding. The resultsare expressed as mean±SE of three independent experiments.

FIG. 5B is a graph showing elution profiles of sCD4 (line 1), S15 (line2), and sCD4-S15 (line 3) from Superdex-200 gel filtration columns (leftpanel). The Gaussian deconvolution of S15-sCD4 and sCD4 peaks is shownin the right panel graph.

FIG. 5C is an immunoblot showing both sCD4 and Salp15 were present inthe same band when preincubated together. Purified sCD4 (10:g), Salp15(10:g) and sCD4 (10:g)+Salp15 (10:g) were subjected to native PAGE andimmunoblotting with anti-CD4 and anti-His antibodies.

FIGS. 6A-6E shows the C-terminal peptide of Salp15 binds CD4 andinhibits T cell activation.

FIG. 6A is a graph showing binding of overlapping synthetic peptides ofSalp15 (0.5:g) to sCD4. The results are representative of fourindependent experiments.

FIG. 6B is a graph showing increasing concentrations of P11 (squares),but not P8 (triangles) show saturable binding to sCD4.

FIG. 6C is a graph showing competition of P11 (50 nmol) binding to sCD4by increasing concentrations of Salp15.

FIG. 6D is a graph showing purified CD4⁺ T cells were activated in vitrowith plate bound anti-CD3 and soluble anti-CD28 in the absence orpresence of Salp15 (50:g/mL), P11, or P8. The supernatants were analyzedat 24 hr of activation for IL-2 levels by capture ELISA.

FIG. 6E shows photomicrographs of CTB₅₉₄ staining in CD4⁺ T cellsstimulated with anti-CD3/CD28 in the absence or presence of Salp15(50:g/mL), P11 (0.2:g/mL), or P8 (0.2:g/mL) for 20 min. The results areexpressed as mean±SE of at least three independent experiments.

FIGS. 7A-7E show Salp15 inhibits early T cell activation events inJurkat cells.

FIG. 7A is a Western blot showing the decrease in anti-CD3/CD28-inducedtyrosine phosphorylation in CD4⁺ T cells (upper panel) in the presenceof Salp15. The middle and lower panels show immunoblotting withanti-actin and anti-Vav1 as loading controls.

FIG. 7B shows representative confocal micrographs of staining withanti-pTyr in Jurkat cells, either unstimulated or stimulated (αCD3/CD28)in the presence or absence of Salp15.

FIG. 7C shows representative confocal micrographs of staining withanti-CTB₅₉₄ in Jurkat cells, either unstimulated or stimulated(αCD3/CD28) in the presence or absence of Salp15.

FIG. 7D shows representative confocal micrographs demonstrating Jurkatcells stimulated with anti-CD3/CD28 in the presence of Salp15 exhibithighly reduced staining with phalloidin₄₈₈ compared to untreatedcontrols.

FIG. 7E is an immunoblot showing the amount of F-actin isolated fromlysates of Jurkat cells either unstimulated or stimulated in thepresence or absence of Salp15 determined by immunoblotting withanti-actin. The lower panel shows the respective amount of total actinin the cell lysates.

FIGS. 8A-8D show Salp15 binds specifically to CD4.

FIG. 8A shows Jurkat cell lysate containing His-tagged Salp15immunoprecipitated using anti-His, anti-CD3ε and anti-CD28. Theimmunoprecipitate was subjected to Western blotting using anti-CD4,anti-CD3ε, anti-CD28 and anti-TCRβ antibodies.

FIG. 8B shows confocal micrographs demonstrating Salp15₄₈₈ binding onthe cell surface of CD4+, but not on CD8⁺ cells.

FIG. 8C shows confocal micrographs of Salp15₄₈₈ binding on HeLa (upperpanels) and HeLa-CD4 cells (lower panels).

FIG. 8D shows immunoprecipitation from HeLa-CD4 cell lysate containingeither His tagged-Salp13-TR fusion protein or Salp15 using anti-Hisfollowed by immunoblotting with anti-CD4 or anti-His antibodies.

FIGS. 9A-9D shows Salp15 specifically inhibits CD4 dependent T cellactivation.

FIG. 9A is a graph showing that the immunosuppressive activity of Salp15was highly diminished in T cells isolated from CD4 deficient mice(filled bars) compared to control T cells (open bars). The results areexpressed as mean±SE of three independent experiments.

FIG. 9B shows Salp15 does not inhibit ERK1/2 phosphorylation in CD4⁺ Tcells activated with anti-CD3/CD28.

FIG. 9C shows Salp15 does not affect STAT1 phosphorylation in CD4⁺ Tcells activated with 20 ng/mL of INFγ.

FIG. 9D is a graph showing Salp15 inhibits IL-2 production in a dosedependent manner in CD4⁺ T cells isolated from DO11.10 transgenic miceactivated with ovalbumin in the presence of APCs from a control mouse.The results are representative of three independent experiments.

FIGS. 10A-10D show the C-terminal peptide of Salp15 binds CD4.

FIG. 10A is a graph showing saturable binding of Salp15 (0.4 μM) withsCD4 D1-D4 (soluble extracellular domains D1-D4 of CD4). Salp15 wasincubated with increasing amounts of immobilized sCD4 D1-D4 (squares) orlysozyme (circle) in a microtiter assay showing.

FIG. 10B is a graph showing competition of increasing concentrations offree P11 with immobilized P11 (50 nmol) for binding to sCD4.

FIG. 100 is a graph showing increasing concentrations of P11-2 (squares)but not PLP (triangles) show saturable binding to sCD4.

FIG. 10D is a graph showing Salp15 treatment with DTT did not affect itsbinding to sCD4-HRP in a microtiter assay. The results are expressed asmean±SE of at least three independent experiments.

FIG. 11 is a graph showing the native gel filtration profile of Salp15.Active and inactive Salp15 was eluted through two superdex-200 gelfiltration columns in tandem. The elution profile of active Salp15(black line) contained predominantly a monomer fraction, while that ofinactive Salp15 (gray line) contained dimer and trimer populations.

FIG. 12 is a graph showing Salp15 effects on CD4-gp120 binding in vitro.

FIG. 13 is a graph showing effects of Salp15 on blocking fusion of cellsexpressing either HIV envelope proteins or CD4.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a polynucleotide sequence encoding a Salp15 polypeptideisolated from Ixodes scapularis.

SEQ ID NO: 2 is a Salp15 polypeptide sequence isolated from Ixodesscapularis.

SEQ ID NO: 3 is a polypeptide fragment sequence (P1) comprising aminoacids 1-20 of SEQ ID NO: 2.

SEQ ID NO: 4 is a polypeptide fragment sequence (P2) comprising aminoacids 11-30 of SEQ ID NO: 2.

SEQ ID NO: 5 is a polypeptide fragment sequence (P3) comprising aminoacids 21-40 of SEQ ID NO: 2.

SEQ ID NO: 6 is a polypeptide fragment sequence (P4) comprising aminoacids 31-50 of SEQ ID NO: 2.

SEQ ID NO: 7 is a polypeptide fragment sequence (P5) comprising aminoacids 41-60 of SEQ ID NO: 2.

SEQ ID NO: 8 is a polypeptide fragment sequence (P6) comprising aminoacids 51-70 of SEQ ID NO: 2.

SEQ ID NO: 9 is a polypeptide fragment sequence (P7) comprising aminoacids 61-80 of SEQ ID NO: 2.

SEQ ID NO: 10 is a polypeptide fragment sequence (P8) comprising aminoacids 71-90 of SEQ ID NO: 2.

SEQ ID NO: 11 is a polypeptide fragment sequence (P9) comprising aminoacids 81-100 of SEQ ID NO: 2.

SEQ ID NO: 12 is a polypeptide fragment sequence (P10) comprising aminoacids 91-110 of SEQ ID NO: 2.

SEQ ID NO: 13 is a polypeptide fragment sequence (P11) comprising aminoacids 95-114 of SEQ ID NO: 2.

SEQ ID NO: 14 is a polypeptide fragment sequence (P11-2) comprisingamino acids 103-114 of SEQ ID NO: 2.

SEQ ID NO: 15 is a polypeptide fragment sequence comprising amino acids139-151 of proteolipid protein 1 (PLP).

SEQ ID NO: 16 is a polynucleotide sequence encoding the polypeptide ofSEQ ID NO: 13.

SEQ ID NO: 17 is a polynucleotide sequence encoding the polypeptide ofSEQ ID NO: 14.

SEQ ID NO: 18 is a forward oligonucleotide primer used to amplify aSalp15 polypeptide by PCR.

SEQ ID NO: 19 is a reverse oligonucleotide primer used to amplify aSalp15 polypeptide by PCR.

DETAILED DESCRIPTION

The presently disclosed subject matter provides compositions capable ofspecifically binding to T-lymphocyte and of modulating Class IIMHC-mediated T-lymphocyte activation via the T cell receptor activationpathway of CD4⁺ T cells and consequently capable of acting asimmunomodulators and anti-inflammatory agents. The compositions havebinding specificity for the T cell CD4 co-receptor and can causeconformational changes to the CD4 co-receptor that can inhibit T cellactivation. The compositions can further act as inhibitors of humanimmunodeficiency virus (HIV) infection of CD4⁺ T cells, as they can bindcompetitively with the CD4 co-receptor and thereby prevent binding ofHIV to the T cell via HIV gp120 polypeptide interaction with T cell CD4.The compositions disclosed herein are further useful in methods ofscreening for additional substances having similar properties as thepresently disclosed compositions.

In some embodiments, the presently disclosed compositions, which arecapable of specifically binding to and modulating CD4⁺ T cells, compriseSalp15, or biologically active fragments thereof. Salp15, disclosedherein for the first time, binds specifically to CD4 on CD4⁺ T cells andacts at the earliest steps of TCR signaling. This can result indiminished tyrosine phosphorylation of effector proteins, defectiveactin polymerization, and/or a reduction in lipid raft reorganization.It has also been discovered, and disclosed herein for the first time,that the immunosuppressive effect of Salp15 can be exerted through adirect and specific association between its C-terminal amino acid regionand the outer two extracellular domains of CD4 (D1-D2). A directassociation between the outer two extracellular domains of CD4 and theC-terminal amino acid residues of Salp15 is sufficient to exert theimmunosuppressive effect of Salp15 on CD4⁺ T cells. Thus, Salp15 andbiologically active fragments thereof, have utility in treating orpreventing with specificity conditions characterized by CD4⁺ T cellresponses, including autoimmune disorders and allogeneic transplanttolerance and as inhibitors of HIV infection of CD4⁺ T cells, asdisclosed in detail herein.

I. General Considerations

Engagement of the T cell receptor complex (TCR) by Class II MHC proteinand associated proteins initiates a complex cascade of biochemicalevents that culminate in proliferation and the initiation of effectorfunctions by T cells. The most proximal and earliest events include theactivation of protein kinases of the Src, Syk, and Tec families (Bolen &Brugge, 1997; Qian & Weiss, 1997), resulting in tyrosine phosphorylationof multiple membrane-bound and cytosolic proteins (Koretzky et al.,2003). The recruitment of these effector proteins to the site ofTCR-Class II MHC interaction, also referred to as the “immunologicalsynapse”, leads to signal amplification resulting in calciummobilization from intracellular stores and interleukin (IL)-2 production(Myung et al., 2000; Zhang & Samelson, 2000), which in turn stimulatesactivation, proliferation, and differentiation of T cells.

Ixodes scapularis salivary protein 15 (Salp15) was recently identifiedas the first antigen responsible for the immunomodulatory action of ticksaliva on acquired immune responses (Anguita et al., 2002, hereinincorporated by reference in its entirety). Salp15 causes the repressionof calcium fluxes triggered by TCR ligation, and therefore NFAT andNF-κB-induced IL-2 transcription. Thus, inhibition of T cell activationmediated by Salp15 results from the repression of calcium fluxestriggered by TCR ligation with a subsequent reduction in IL-2production.

I. scapularis ticks act as the vector for several pathogens includingthe causative agents of Lyme disease and human granulocytic ehrlichiosis(Burgdorfer et al., 1982; Chen et al., 1994). In order to thrive innature, ticks are able to modulate the host immune response. Ticksalivary proteins enter the host during feeding and exert pleiotropicimmunosuppressive effects (Anguita et al., 2002; Ferreira & Silva, 1998;Kopecky & Kuthejlova, 1998; Ribeiro et al., 1995; Schoeler et al., 1999;Urioste et al., 1994; Wikel & Bergman, 1997). Immunosuppression of thehost by tick saliva could also contribute to the efficient transmissionof tick-borne pathogens (Wikel, 1999 & Ramamoorthi et al., 2005, hereinincorporated by reference).

Salp15 is a candidate for use in immunosuppressive therapies. A clearunderstanding of the mechanism by which Salp15 causes immunosuppression,however, is a prerequisite for continuing further studies regarding itspotential use. Prior to the discovery of the subject matter disclosedherein, a full understanding of Salp15 mechanism of T cell inhibitionwas unknown.

II. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage canencompass variations of in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

The term “antibody” or “antibody molecule” refers collectively to apopulation of immunoglobulin molecules and/or immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain aparatope. A paratope is the portion or portions of antibodies that is orare responsible for that antibody binding to an antigenic determinant,or epitope.

Representative antibodies for use in the present subject matter areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules, single chain immunoglobulins or antibodies, those portions ofan immunoglobulin molecule that contain the paratope, including antibodyfragments. A monovalent antibody can optionally be used.

The terms “associated with”, “operably linked”, and “operatively linked”refer to two nucleic acid sequences that are related physically orfunctionally. For example, a promoter or regulatory DNA sequence is saidto be “associated with” a DNA sequence that encodes an RNA or apolypeptide if the two sequences are operatively linked, or situatedsuch that the regulator DNA sequence will affect the expression level ofthe coding or structural DNA sequence.

The terms “CD4”, “CD4 receptor”, and “CD4 co-receptor” are usedinterchangeably herein and refer to a transmembrane glycoproteinexpressed on certain immune system cells, including helper T cells (CD4⁺T cells). CD4 expressed on helper T cells acts as a co-receptor alongwith the T cell receptor complex in the TCR-mediated activation pathwayof T cells, which occurs when a TCR (along with accessory molecules,including CD4) recognizes and binds to, in the case of helper T cells,an MHC Class II molecule presenting a particular antigen recognized bythe TCR. After initial binding of TCR with the Class II MHCmolecule-antigen complex, CD4 binds the Class II MHC molecule at a siteseparate from the TCR binding site. CD4 primarily functions as part ofthe T cell activation signaling cascade, but might also play a role inadhesion to stabilize the TCR-MHC complex. CD4 is expressed as a monomerwith four extracellular Ig-like domains, numbered D1 through D4beginning from the N-terminus (D1-D4) (Capon et al., 1989; Fleury etal., 1991), a hydrophobic transmembrane domain, and a highly basiccytoplasmic tail of 38 amino acids. CD4 binds Class II MHC through itstwo N-terminal extracellular domains (D1-D2).

As used herein, “CD4” refers not only to the full-length protein, butbiologically active fragments as well. In particular, the extracellularfour domains of CD4 (sCD4) or even D1-D2 alone can function to bindClass II MHC, and so these fragments are biologically active in thesense of binding MHC and may be referred to herein simply as CD4, forexample. Further, Salp15 can specifically bind the D1-D2 fragment of CD4and so is a particularly relevant biologically active fragment for thepurposes of the presently disclosed subject matter.

The terms “coding sequence” and “open reading frame” (ORF) are usedinterchangeably and refer to a nucleic acid sequence that is transcribedinto RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA.In some embodiments, the RNA is then translated in vivo or in vitro toproduce a polypeptide.

The term “complementary” refers to two nucleotide sequences thatcomprise antiparallel nucleotide sequences capable of pairing with oneanother upon formation of hydrogen bonds between the complementary baseresidues in the antiparallel nucleotide sequences. As is known in theart, the nucleic acid sequences of two complementary strands are thereverse complement of each other when each is viewed in the 5′ to 3′direction.

The term “fragment” refers to a sequence that comprises a subset ofanother sequence. When used in the context of a nucleic acid or aminoacid sequence, the terms “fragment” and “subsequence” are usedinterchangeably. A fragment of a nucleic acid sequence can be any numberof nucleotides that is less than that found in another nucleic acidsequence, and thus includes, but is not limited to, the sequences of anexon or intron, a promoter, an enhancer, an origin of replication, a 5′or 3′ untranslated region, a coding region, and a polypeptide bindingdomain. It is understood that a fragment or subsequence can alsocomprise less than the entirety of a nucleic acid sequence, for example,a portion of an exon or intron, promoter, enhancer, etc. Similarly, afragment or subsequence of an amino acid sequence can be any number ofresidues that is less than that found in a naturally occurringpolypeptide, and thus includes, but is not limited to, domains,features, repeats, etc. Also similarly, it is understood that a fragmentor subsequence of an amino acid sequence need not comprise the entiretyof the amino acid sequence of the domain, feature, repeat, etc.

A fragment can also be a “functional fragment”, in which the fragmentretains a specific biological function of the nucleic acid sequence oramino acid sequence of interest. For example, a functional fragment of aSalp15 polypeptide can include a region having binding specificity for aCD4 co-receptor and/or capable of modulating activation of a CD4⁺ Tcell.

The term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include, but are notlimited to, coding sequences and/or the regulatory sequences requiredfor their expression. Genes can also include non-expressed DNA segmentsthat, for example, form recognition sequences for a polypeptide. Genescan be obtained from a variety of sources, including cloning from asource of interest or synthesizing from known or predicted sequenceinformation, and can include sequences designed to have desiredparameters.

The terms “heterologous”, “recombinant”, and “exogenous”, when usedherein to refer to a nucleic acid sequence (e.g. a DNA sequence) or agene, refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modifiedthrough, for example, the use of site-directed mutagenesis or otherrecombinant techniques. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position or form within the host cell inwhich the element is not ordinarily found. Similarly, when used in thecontext of a polypeptide or amino acid sequence, an exogenouspolypeptide or amino acid sequence is a polypeptide or amino acidsequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, exogenous DNA segments can be expressed to yield exogenouspolypeptides.

A “homologous” nucleic acid (or amino acid) sequence is a nucleic acid(or amino acid) sequence naturally associated with a host cell intowhich it is introduced.

The term “inhibitor” refers to a chemical substance that inactivates ordecreases the biological activity of a target entity such as acomplement component.

The term “isolated”, when used in the context of an isolated DNAmolecule or an isolated polypeptide, is a DNA molecule or polypeptidethat, by the hand of man, exists apart from its native environment andis therefore not a product of nature. An isolated DNA molecule orpolypeptide can exist in a purified form or can exist in a non-nativeenvironment such as, for example, in a transgenic host cell.

The term “MHC polypeptide” and “MHC molecule” as used herein refers to apolypeptide expressed on the membrane surface of an antigen presentingcell (APC), which serves as a peptide display (antigen presentation)molecule for recognition by T cells, and more specifically, by the TCRsin complex with other signaling molecules of the T cells. MHCpolypeptides can generally be categorized into two structurally distinctclasses. Class I MHC molecules are present on most nucleated cells, bindand present peptides derived from cytosolic proteins, and are recognizedby TCRs on CD8⁺ T cells. Class II MHC polypeptides are restrictedprimarily to “professional” antigen presenting cells (such asmacrophages and dendritic cells), bind and present peptides derived fromendocytosed proteins, and are recognized by TCRs on CD4⁺ T cells.

As used herein, the term “modulate” means an increase, decrease, orother alteration of any, or all, chemical and biological activities orproperties of a target entity, such as a wild-type or mutantpolypeptide, including but not limited to a Salp15 polypeptide. The term“modulation” as used herein refers to both upregulation (i.e.,activation or stimulation) and downregulation (i.e., inhibition orsuppression) of a response, such as for example, modulation of a T cellactivation response, including modulation of activation andproliferation of the T cell resulting from the modulated response.

As used herein, the term “mutation” carries its traditional connotationand means a change, inherited, naturally occurring or introduced, in anucleic acid or polypeptide sequence, and is used in its sense asgenerally known to those of skill in the art.

As used herein, the terms “T lymphocyte” or “T cell” are usedinterchangeably and refer to cells of the immune system that mediatecell-mediated immune responses as part of the adaptive immune system. Tcells are named for their tissue of origin, the thymus. T cells,however, circulate in the blood, populate lymphoid tissues and somesubclasses are recruited to peripheral sites of antigen exposure whenactivated to facilitate an immune response to an antigen. T cellsexpress T cell receptors (TCRs) that recognize antigen (e.g., peptidefragments) when bound to MHC molecules. T cells can be further dividedinto subclasses of CD4⁺ T cells (referred to as helper T cells) and CD8⁺T cells (referred to as cytotoxic T lymphocytes), which express asmembrane bound co-receptors of TCR CD4 or CD8, respectively.

As used herein, the term “T cell receptor” or “TCR” refers to a clonallydistributed polypeptide expressed on the membrane surface of CD4⁺ andCD8⁺ T lymphocytes. TCRs are antigen receptors that function as acomponent of the immune system for recognition of peptides bound to MHCmolecules on the surface of antigen presenting cells. A TCR comprises adiversity or variable region within its polypeptide sequence thatcontributes to the determination of the particular antigen and MHCmolecule to which the TCR has binding specificity. In turn, thespecificity of a T cell for a unique antigen-MHC complex resides in theparticular TCR expressed by the T cell.

The most common structural form of a TCR found in vivo is as aheterodimer of two disulfide-linked transmembrane polypeptide chains,designated α and β, each chain comprising one N-terminal diversityregion, one immunoglobulin-like constant domain, a hydrophobictransmembrane region and a short cytoplasmic region. A less common typeof TCR comprising γ and δ chains is found in a small subset of cells andis included by the term TCR as used herein.

Although the term TCR, as used herein, includes the entire heterodimerstructure, the term is not intended to be limited to this singledefinition. TCR, as used herein, further includes each α and β chainindividually, as well as biologically active fragments thereof,including fragments soluble in aqueous solutions, of either chain aloneor both chains joined. Biologically active fragments maintain theability to at least bind with specificity to a specific antigen, andtherefore will include at least a portion of the diversity regionimparting antigen-MHC complex specificity. Biologically active fragmentsof TCRs disclosed herein can further include other functionalities offull-length TCRs, such as forming a TCR complex with other proteins,such as signaling proteins, on the membrane surface of a T cell andactivating the T cell. TCR as used herein also includes, and is usedinterchangeably with the term “TCR complex”, which comprises the TCRalong with non-covalently associated proteins that play a role intransduction of the signal arising from TCR binding to a particularantigen-MHC complex, such as for example CD3 and ξ proteins.

TCRs normally play a role in recognition of foreign antigens, followedby T cell activation, proliferation (i.e., clonal expansion of theactivated T cell), and differentiation (i.e., maturation to either aneffector or memory T cell, each having distinct roles in the immunesystem) with a resultant activation and targeting of the immune systemagainst the foreign antigen. However, TCRs can sometimes havespecificity for and activate when contacted with MHC presentedself-antigens, also referred to herein as autoantigens. Activation of Tcells as a result of binding by TCRs to autoantigen-MHC complexes canplay a role in certain autoimmune diseases, including for examplemultiple sclerosis. T cell activation via TCR-mediated signaling alsoplays a role in tissue and organ transplant rejection.

The term “transformation” refers to a process for introducingheterologous DNA into a cell. Transformed cells are understood toencompass not only the end product of a transformation process, but alsotransgenic progeny thereof.

The terms “transformed”, “transgenic”, and “recombinant” refer to a cellof a host organism such as a mammal into which a heterologous nucleicacid molecule has been introduced. The nucleic acid molecule can bestably integrated into the genome of the cell or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or subjects are understood to encompass not only the endproduct of a transformation process, but also transgenic progenythereof. A “non-transformed,” “non-transgenic”, or “non-recombinant”host refers to a wild type organism, e.g., a mammal or a cell therefrom,which does not contain the heterologous nucleic acid molecule.

III. Polypeptides and Nucleic Acids

The presently disclosed subject matter discloses isolated and purifiedbiologically active Salp15 polypeptides and nucleic acid moleculesencoding same. As used in the following detailed description and in theclaims, the term “Salp15” includes tick (e.g. Ixodes scapularis)salivary protein 15 polypeptides, and biologically functionalequivalents thereof and nucleic acids encoding same. The term “Salp15”includes homologs from non-tick species. Preferably, Salp15 nucleicacids and polypeptides are isolated from eukaryotic sources.

The terms “Salp15 gene product”, “Salp15 protein”, and “Salp15polypeptide” refer to peptides having amino acid sequences which aresubstantially identical to native amino acid sequences from the organismof interest and which are biologically active in that they comprise allor a part of the amino acid sequence of a Salp15 protein, or cross-reactwith antibodies raised against a Salp15 polypeptide, or retain all orsome of the biological activity of the native amino acid sequence orprotein. For example, in one embodiment a Salp15 protein is apolypeptide isolated originally as a secreted salivary protein fromIxodes scapularis and set forth herein as SEQ ID NO: 2 and encoded by apolynucleotide, for example, as set forth in SEQ ID NO:1. See alsoAnguita et al., 2002.

In some embodiments, the Salp15 polypeptide is modified to be in adetectably labeled form. A labeled form of the Salp15 polypeptide hasseveral utilities, as would be appreciated by one of skill in the art.For example, a labeled Salp15 polypeptide could be used to identify thepresence of a molecule to which Salp15 binds with specificity in asample, e.g., a CD4 receptor polypeptide. The molecule to which Salp15binds could be soluble or bound. For example, the molecule could beexpressed by a cell, or certain types of cells, and a labeled Salp15polypeptide could be utilized to determine whether a population ofcells, or individual members thereof, express the molecule. Methods ofusing a labeled Salp15 polypeptide in this manner are known to those ofskill in the art. For example, a population of cells could be quicklyscreened for cells expressing a molecule to which Salp15 binds withspecificity (e.g., CD4) using a labeled Salp15 polypeptide inconjunction with a fluorescence activated cell sorter.

The terms “Salp15 gene product”, “Salp15 protein”, and “Salp15polypeptide” also include biologically functional equivalents andanalogs of Salp15. By “analog” is intended that a DNA or peptidesequence can contain alterations relative to the sequences disclosedherein, yet retain all or some of the biological activity of thosesequences. Analogs can be derived from genomic nucleotide sequences asare disclosed herein or from other organisms, or can be createdsynthetically. Those skilled in the art will appreciate that otheranalogs, as yet undisclosed or undiscovered, can be used to designand/or construct Salp15 analogs. There is no need for a “Salp15 geneproduct”, “Salp15 protein”, and “Salp15 polypeptide” to comprise all orsubstantially all of the amino acid sequence of a native Salp15 geneproduct. Shorter or longer sequences are anticipated to be of use in thepresently disclosed subject matter; shorter sequences are hereinreferred to as “fragments” or “segments”. Thus, the terms “Salp15 geneproduct”, “Salp15 protein”, and “Salp15 polypeptide” also includefragment, fusion, chemically modified, or recombinant Salp15polypeptides and proteins comprising sequences of the presentlydisclosed subject matter. Methods of preparing such proteins are knownin the art.

The terms “Salp15 gene”, “Salp15 gene sequence”, and “Salp15 genefragment” refer to any DNA sequence that is substantially identical to apolynucleotide sequence encoding a Salp15 gene product, protein orpolypeptide as defined above, and can also comprise any combination ofassociated control sequences. The terms also refer to RNA, or antisensesequences, complementary to such DNA sequences. As used herein, the term“DNA segment” or “DNA fragment” refers to a DNA molecule that has beenisolated free of total genomic DNA of a particular species. Furthermore,a DNA segment encoding a Salp15 polypeptide refers to a DNA segment thatcontains Salp15 coding sequences, yet is isolated away from, or purifiedfree from, total genomic DNA of a source species, such as I. scapularis.Included within the term “DNA segment” are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phages, viruses, and the like.

Exemplary polypeptide fragments of Salp15 encompassed by the presentlydisclosed subject matter are set forth in Table 1 (SEQ ID NOs: 3-14; SEQID NO: 15 is a non-SALP15 polypeptide negative control). As disclosedherein in the EXAMPLES, Salp15 polypeptide fragments P11 (SEQ ID NO: 13)and P11-2 (SEQ ID NO: 14) are derived from the C-terminus of Salp15 andhave been experimentally determined to exhibit the highest activity ofthe tested polypeptide fragments of Salp15 for modulating naïve CD4⁺ Tcell activation. SEQ ID NOs: 16 and 17 set forth the polynucleotidecoding sequences for P11 and P11-2, respectively.

TABLE 1 Overlapping Synthetic Peptides of Salp15 Peptide PositionSequence SEQ. ID NO: P1   1-20 NESGPTKADASTADKDTKKN 3 P2  11-30STADKDTKKNNVQLRFPNYI 4 P3  21-40 NVQLRFPNYISNHQKLALKL 5 P4  31-50SNHQKLALKLLKICKDSKSS 6 P5  41-60 LKICKDSKSSHNSLSSRSSD 7 P6  51-70HNSLSSRSSDVINDKYVDFK 8 P7  61-80 VINDKYVDFKNCTFLCKHGN 9 P8  71-90NCTFLCKHGNDVNVTLNLPE 10 P9  81-100 DVNVTLNLPEDTPCGPNGQT 11 P10  91-110DTPCGPNGQTCAEKNKCVGH 12 P11*  95-114 GPNGQTCAEKNKCVGHIPGC 13 P11-2*103-114 EKNKCVGHIPGC 14 PLP 139-151 HSLGKWLGHPDKF 15 *Polynucleotidecoding sequences for: P11: gga ccg aat gga cag aca tgc gct gaa aag aataaa tgc gtt ggc cac att ccc gga tgt (SEQ ID NO: 16); and P11-2: gaa aagaat aaa tgc gtt ggc cac att ccc gga tgt (SEQ ID NO: 17).

The term “substantially identical”, when used to define either a Salp15gene product or amino acid sequence, or a Salp15 gene or nucleic acidsequence, means that a particular sequence varies from the sequence of anatural Salp15 or fragment thereof by one or more deletions,substitutions, or additions, the net effect of which is to retain atleast some of the biological activity of the natural gene, gene product,or sequence. Such sequences include “mutant” sequences, or sequences inwhich the biological activity is altered to some degree but retains atleast some of the original biological activity.

Alternatively, DNA analog sequences are “substantially identical” tospecific DNA sequences disclosed herein if: (a) the DNA analog sequenceis derived from coding regions of the natural Salp15 gene; or (b) theDNA analog sequence is capable of hybridization of DNA sequences of (a)under stringent conditions and which encode biologically active Salp15gene product; or (c) the DNA sequences are degenerate as a result ofalternative genetic code to the DNA analog sequences defined in (a)and/or (b). Substantially identical analog proteins will be greater thanabout 80% identical to the corresponding sequence of the native proteinor biologically active fragment thereof. Sequences having lesser degreesof identity but comparable biological activity are considered to beequivalents. In determining nucleic acid sequences, all subject nucleicacid sequences capable of encoding substantially similar amino acidsequences are considered to be substantially similar to a referencenucleic acid sequence, regardless of differences in codon sequences orsubstitution of equivalent amino acids or modifications to amino acids(e.g., chemical modifications) to create biologically functionalequivalents.

Sequence identity or percent similarity of a DNA or peptide sequence canbe determined, for example, by comparing sequence information using theGAP computer program, available from the University of WisconsinGeneticist Computer Group. The GAP program utilizes the alignment methodof Needleman et al., 1970, as revised by Smith et al., 1981. Briefly,the GAP program defines similarity as the number of aligned symbols(i.e., nucleotides or amino acids) that are similar, divided by thetotal number of symbols in the shorter of the two sequences. Thepreferred parameters for the GAP program are the default parameters,which do not impose a penalty for end gaps. See Schwartz et al., 1979;Gribskov et al., 1986.

In certain embodiments, the presently disclosed subject matter concernsthe use of Salp15 genes and gene products that include within theirrespective sequences a sequence that is essentially that of a Salp15gene, or the corresponding protein, or fragments thereof. The term “asequence essentially as that of a Salp15 gene”, means that the sequenceis substantially identical or substantially similar to a portion of aSalp15 gene or gene products and contains a minority of bases or aminoacids (whether DNA or protein) which are not identical to those of aSalp15 protein or a Salp15 gene, or which are not a biologicallyfunctional equivalent. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein.Nucleotide sequences are “essentially the same” where they have betweenabout 80% and about 85% or more preferably, between about 86% and about90%, or more preferably greater than 90%, or more preferably about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; of nucleic acid residueswhich are identical to the nucleotide sequence of a Salp15 gene.Similarly, peptide sequences which have about 80%, or 90% or greater, orabout 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater aminoacids which are identical or functionally equivalent or biologicallyfunctionally equivalent to the amino acids of a Salp15 polypeptide willbe sequences which are “essentially the same”.

Salp15 gene products and Salp15 encoding nucleic acid sequences, whichhave functionally equivalent codons, are also covered by the subjectmatter disclosed herein. The term “functionally equivalent codon” isused herein to refer to codons that encode the same amino acid, such asthe ACG and AGU codons for serine. Thus, when referring to the sequenceexamples presented in SEQ ID NOs: 1, 16 or 17, for example, applicantscontemplate substitution of functionally equivalent codons of Table 2into the sequence examples of SEQ ID NOs: 1, 16 or 17. Thus, applicantsare in possession of amino acid and nucleic acid sequences which includesuch substitutions but which are not set forth herein in their entiretyfor convenience.

TABLE 2 Functionally Equivalent Codons Amino Acids Codons Alanine Ala AGCA; GCC; GCG; GCU Cysteine Cys C UGC; UGU Aspartic Acid Asp D GAC; GAUGlutamic acid Glu E GAA; GAG Phenylalanine Phe F UUC; UUU Glycine Gly GGGA; GGC; GGG; GGU Histidine His H CAC; CAU Isoleucine Ile I AUA; AUC;AUU Lysine Lys K AAA; AAG Leucine Leu L UUA; UUG; CUA; CUC; CUG; CUUMethionine Met M AUG Asparagine Asn N AAC; AAU Proline Pro P CCA; CCC;CCG; CCU Glutamine Gln Q CAA; CAG Arginine Arg R AGA; AGG; CGA; CGC;CGG; CGU Serine Ser S ACG; AGU; UCA; UCC; UCG; UCU Threonine Thr T ACA;ACC; ACG; ACU Valine Val V GUA; GUC; GUG; GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC; UAU

It will also be understood by those of skill in the art that amino acidand nucleic acid sequences can include additional residues, such asadditional N- or C-terminal amino acids or 5′ or 3′ nucleic acidsequences, and yet still be essentially as set forth in one of thesequences disclosed herein, so long as the sequence retains biologicalprotein activity where protein expression is concerned. The addition ofterminal sequences particularly applies to nucleic acid sequences whichcan, for example, include various non-coding sequences flanking eitherof the 5′ or 3′ portions of the coding region or can include variousinternal sequences, i.e., introns, which are known to occur withingenes.

The present subject matter also encompasses the use of nucleotidesegments that are complementary to the sequences of the presentlydisclosed subject matter, in one embodiment, segments that are fullycomplementary, i.e., complementary for their entire length. Nucleic acidsequences that are “complementary” are those, which are base-pairedaccording to the standard Watson-Crick complementarity rules. As usedherein, the term “complementary sequences” means nucleic acid sequenceswhich are substantially complementary, as can be assessed by the samenucleotide comparison set forth above, or is defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein. A particularexample of a complementary nucleic acid segment is an antisenseoligonucleotide.

One technique in the art for assessing complementary sequences and/orisolating complementary nucleotide sequences is hybridization. Nucleicacid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess ofabout 30° C., typically in excess of about 37° C., and preferably inexcess of about 45° C. Stringent salt conditions will ordinarily be lessthan about 1,000 mM, typically less than about 500 mM, and preferablyless than about 200 mM. However, the combination of parameters is muchmore important than the measure of any single parameter. See e.g.,Wethmur & Davidson, 1968. Determining appropriate hybridizationconditions to identify and/or isolate sequences containing high levelsof homology is well known in the art. See e.g., Sambrook et al., 2001.

For the purposes of specifying conditions of high stringency, preferredconditions are salt concentration of about 200 mM and temperature ofabout 45° C. One example of such stringent conditions is hybridizationat 4×SSC, at 65° C., followed by a washing in 0.1×SSC at 65° C. for onehour. Another exemplary stringent hybridization scheme uses 50%formamide, 4×SSC at 42° C. Another example of “stringent conditions”refers to conditions of high stringency, for example 6×SSC, 0.2%polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1%sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA and 15% formamide at68° C. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 10×SSC (0.9 M NaCl/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1×SSC. Sequence identity can bedetermined by hybridization under stringent conditions, for example, at50° C. or higher and 0.1×SSC (9 mM NaCl/0.9 mM sodium citrate).

Nucleic acids that are substantially identical to the provided Salp15sequences, e.g., allelic variants, genetically altered versions of thegene, etc., bind to the provided Salp15 sequences under stringenthybridization conditions. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes. Thesource of homologous genes can be any species, e.g., arthropod species,particularly tick species (Order acari), and also including primatespecies, particularly human; rodents, such as rats and mice, canines,felines, bovines, ovines, equines, yeast, nematodes, etc.

Between arthropod species, e.g., ticks, mites, insects, and spiders,homologs have substantial sequence similarity, i.e., at least 80%sequence identity between nucleotide sequences. Sequence similarity iscalculated based on a reference sequence, which can be a subset of alarger sequence, such as a conserved motif, coding region, flankingregion, etc. A reference sequence will usually be at least about 18nucleotides long, more usually at least about 30 nucleotides long, andcan extend to the complete sequence that is being compared. Algorithmsfor sequence analysis are known in the art, such as BLAST, described inAltschul et al., 1990. The sequences provided herein are essential forrecognizing Salp15 related and homologous proteins in database searches.

At a biological level, identity is just that, i.e., the same amino acidat the same relative position in a given family member of a gene family.Homology and similarity are generally viewed as broader terms. Forexample, biochemically similar amino acids, for example leucine andisoleucine or glutamate/aspartate, can be present at the sameposition—these are not identical per se, but are biochemically“similar”. As disclosed herein, these are referred to as conservativedifferences or conservative substitutions. This differs from aconservative mutation at the DNA level, which changes the nucleotidesequence without making a change in the encoded amino acid, e.g., TCC toTCA, both of which encode serine.

The Salp15 polypeptides disclosed herein are thus homologouspolypeptides, but when percentages are referred to herein, it is meantto refer to percent identity. The percent identities referenced hereincan be generated, for example, by alignments with the program GENEWORKS™(Oxford Molecular, Inc. of Campbell, Calif., United States of America)and/or the BLAST program at the NCBI website. Another commonly usedalignment program is entitled CLUSTAL W and is described in Thompson etal., 1994, among other places.

Probe sequences can also hybridize specifically to duplex DNA undercertain conditions to form triplex or other higher order DNA complexes.The preparation of such probes and suitable hybridization conditions aredisclosed herein and are known in the art.

The term “gene” is used for simplicity to refer to a functional protein,polypeptide or peptide encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences and cDNAsequences. Preferred embodiments of genomic and cDNA sequences aredisclosed herein.

In particular embodiments, the presently disclosed subject matterconcerns isolated DNA segments and recombinant vectors incorporating DNAsequences, which encode a Salp15 polypeptide or biologically activefragment thereof that includes within its amino acid sequence an aminoacid sequence as described herein. In other particular embodiments, thepresently disclosed subject matter concerns recombinant vectorsincorporating DNA segments, which encode a protein comprising the aminoacid sequence of an I. scapularis Salp15 protein (for example, but notlimited to SEQ ID NO: 2) or biologically functional equivalents thereof(for example, but not limited to SEQ ID NOs: 13 and 14).

III.A. Biologically Functional Equivalents

As mentioned above, modifications and changes can be made in thestructure of the Salp15 proteins and peptide fragments described hereinand still constitute a molecule having like or otherwise desirablecharacteristics. For example, certain amino acids can be substituted forother amino acids or chemically modified (e.g., to increase stability ofthe peptide) in a protein structure without appreciable loss ofinteractive capacity with, for example, proteins expressed on thesurface of T cells, including in particular the CD4 co-receptorexpressed by helper T cells (T_(H)) cells, which can modulate activationof these T cells. Since it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence modifications or substitutions can be madein a protein sequence (or the nucleic acid sequence encoding it) toobtain a protein with the same, enhanced, or antagonistic properties.Such properties can be achieved by interaction with the normal targetsof the native protein, but this need not be the case. It is thusprovided in accordance with the present subject matter that variousmodifications or changes can be made in the sequence of the Salp15proteins and peptides or underlying nucleic acid sequence withoutappreciable loss of their biological utility or activity.

Biologically functional equivalent peptides, as used herein, arepeptides in which certain, but not most or all, of the amino acids canbe substituted and/or chemical modifications, substitutions or additionsare made to one or more amino acids. Thus, for example, when referringto the sequence examples presented in SEQ ID NOs: 2, 13, and 14applicants contemplate substitution of codons that encode biologicallyequivalent amino acids as described herein into the sequence examples ofSEQ ID NOs: 2, 13, and 14. Thus, applicants are in possession of aminoacid and nucleic acids sequences which include such substitutions butwhich are not set forth herein in their entirety for convenience.

Alternatively, functionally equivalent proteins or peptides can becreated via the application of recombinant DNA technology, in whichchanges in the protein structure can be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man can be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein or to test Salp15 mutants in order toexamine Salp15 activity at the molecular level.

Amino acid substitutions, such as those which might be employed inmodifying the Salp15 proteins and peptides described herein, aregenerally based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. An analysis of the size, shape and type of the aminoacid side-chain substituents reveals that arginine, lysine and histidineare all positively charged residues; that alanine, glycine and serineare all of similar size; and that phenylalanine, tryptophan and tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas biologically functional equivalents. Those of skill in the art willappreciate other biologically functionally equivalent changes.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982, incorporated herein by reference). It isknown that certain amino acids can be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,the substitution of amino acids whose hydropathic indices are within ±2of the original value is preferred, those, which are within ±1 of theoriginal value, are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e., with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those, which are within ±1 of theoriginal value, are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges can be effected by alteration of the encoding DNA, taking intoconsideration also that the genetic code is degenerate and that two ormore codons can code for the same amino acid.

Thus, it will also be understood that the presently disclosed subjectmatter is not limited to the particular nucleic acid and amino acidsequences of SEQ ID NOs: 1, 2, 13, 14, 16, and 17. Recombinant vectorsand isolated DNA segments can therefore variously include the Salp15polypeptide-encoding region itself, include coding regions bearingselected alterations or modifications in the basic coding region, orinclude larger polypeptides which nevertheless comprise Salp15-encodingregions or can encode biologically functional equivalent proteins orpeptides which have variant amino acid sequences, or can encodebiologically functional equivalent fragments of the entire Salp15,including in particular fragments of the C-terminus of Salp15 havingbinding specificity for T cell-expressed proteins, including CD4co-receptor. Biological activity of a Salp15 polypeptide can includebinding specificity for CD4 co-receptor and ability to modulateactivation of T cells. Determining biological activity as describedherein is within the ordinary skill of one skilled in the art, uponreview of the present disclosure. Exemplary procedures for determiningbiological activity of Salp15 polypeptides are disclosed herein in theEXAMPLES.

In particular embodiments, the presently disclosed subject matterconcerns isolated DNA sequences and recombinant DNA vectorsincorporating DNA sequences that encode a protein comprising the aminoacid sequence of the Salp15 polypeptide from I. scapularis. In certainother embodiments, the present subject matter concerns isolated DNAsegments and recombinant vectors that comprise a nucleic acid sequenceessentially as set forth in SEQ ID NOs: 16 or 17.

The nucleic acid segments of the present subject matter, regardless ofthe length of the coding sequence itself, can be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length can be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments can beprepared which include a short stretch complementary to a nucleic acidsequence set forth in any of SEQ ID NOs: 1, 16, or 17 such as about 10nucleotides, and which are up to 10,000 or 5,000 base pairs in length,with segments of 3,000 being preferred in certain cases. DNA segmentswith total lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100,and about 50 base pairs in length are also contemplated to be useful.

The DNA segments of the present subject matter encompass biologicallyfunctionally equivalent Salp15 proteins and peptides. Such sequences canarise as a consequence of codon redundancy and functional equivalencythat are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides can be created via the application of chemical synthesis orrecombinant DNA technology, in which changes in the protein structurecan be engineered, based on considerations of the properties of theamino acids being exchanged. Changes can be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test Salp15mutants in order to examine activity in the modulation of, for example,binding specificity for CD4 co-receptor polypeptides, modulation of Tcell activity, inhibition of HIV infection, or other activity at themolecular level. Site-directed mutagenesis techniques are known to thoseof skill in the art and are disclosed herein.

The presently disclosed subject matter further encompasses fusionproteins and peptides wherein the Salp15 coding region is aligned withinthe same expression unit with other proteins or peptides having desiredfunctions, such as for purification, labeling, or immunodetectionpurposes.

Recombinant vectors form further aspects of the present disclosure.Particularly useful vectors are those in which the coding portion of theDNA segment is positioned under the control of a promoter. The promotercan be that naturally associated with the Salp15 gene, as can beobtained by isolating the 5′ non-coding sequences located upstream ofthe coding segment or exon, for example, using recombinant cloningand/or polymerase chain reaction (PCR) technology and/or other methodsknown in the art, in conjunction with the compositions disclosed herein.

In other embodiments, it is provided that certain advantages will begained by positioning the coding DNA segment under the control of, i.e.,operatively linked to, a recombinant, or heterologous, promoter. As usedherein, a recombinant or heterologous promoter is a promoter that is notnormally associated with a Salp15 gene in its natural environment. Suchpromoters can include promoters isolated from bacterial, viral,eukaryotic, or mammalian cells. Naturally, it will be important toemploy a promoter that effectively directs the expression of the DNAsegment in the cell type chosen for expression. The use of promoter andcell type combinations for protein expression is generally known tothose of skill in the art of molecular biology (See e.g., Sambrook etal., 2001). The promoters employed can be constitutive or inducible andcan be used under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides. Appropriatepromoter systems contemplated for use in high-level expression include,but are not limited to, the vaccinia virus promoter and the baculoviruspromoter.

In an alternative embodiment, the presently disclosed subject matterprovides an expression vector comprising a polynucleotide that encodes abiologically active Salp15 polypeptide in accordance with the presentdisclosure. In one example, an expression vector of the present subjectmatter comprises a polynucleotide that encodes a Salp15 gene product. Inanother example, an expression vector of the present subject mattercomprises a polynucleotide that encodes a polypeptide comprising anamino acid residue sequence of SEQ ID NOs: 13 or 14. In yet anotherexample, an expression vector of the presently disclosed subject mattercomprises a polynucleotide comprising the nucleotide sequence of SEQ IDNOs: 16 or 17. Optionally, an expression vector of the presentlydisclosed subject matter comprises a polynucleotide operatively linkedto an enhancer-promoter. For example, an expression vector can comprisea polynucleotide operatively linked to a prokaryotic promoter.Alternatively, an expression vector of the presently disclosed subjectmatter comprises a polynucleotide operatively linked to anenhancer-promoter that is a eukaryotic promoter and the expressionvector further comprises a polyadenylation signal that is positioned 3′of the carboxy-terminal amino acid and within a transcriptional unit ofthe encoded polypeptide.

In yet another embodiment, disclosed herein is a recombinant host celltransfected with a polynucleotide that encodes a biologically activeSalp15 polypeptide in accordance with the present subject matter. SEQ IDNOs: 16 and 17 and 13 and 14 set forth representative nucleotide andamino acid sequences of Salp15, respectively, from ticks. Also providedare homologous or biologically functionally equivalent polynucleotidesand Salp15 polypeptides found in other animals, including for exampleother arthropod homologs. Optionally, a recombinant host cell of thepresent subject matter is transfected with the polynucleotide thatencodes a Salp15 polypeptide. As another option, a recombinant host cellis transfected with the polynucleotide sequence encoding or set forth inSEQ ID NOs: 16 or 17. A recombinant host cell is a bacterial cell, amammalian cell or an insect cell. In some embodiments, the host cell isan attenuated bacterium, such as for example, attenuated Salmonella andthe host is utilized to deliver the Salp15 polynucleotide sequence to atarget cell or tissue within a subject, wherein the Salp15 polypeptideis translated from the polynucleotide. Motameni et al., 2004 disclosesrepresentative methods for engineering the exemplary attenuatedSalmonella host cells, and is incorporated herein by reference in itsentirety.

In another aspect, a recombinant host cell is a prokaryotic host cell,including parasitic and bacterial cells. Preferably, a recombinant hostcell is a bacterial cell, for example, a strain of Escherichia coli. Therecombinant host cell can comprise a polynucleotide under thetranscriptional control of regulatory signals functional in therecombinant host cell, wherein the regulatory signals appropriatelycontrol expression of the Salp15 polypeptide in a manner to enable allnecessary transcriptional and post-transcriptional modification.

In yet another embodiment, provided is a process of preparing a Salp15polypeptide comprising transfecting a cell with polynucleotide thatencodes a biologically active Salp15 polypeptide as disclosed herein, toproduce a transformed host cell, and maintaining the transformed hostcell under biological conditions sufficient for expression of thepolypeptide. The polypeptide can be isolated if desired, using anysuitable technique. The host cell can be a prokaryotic or eukaryoticcell, such as, but not limited to a bacterial cell of Salmonella sp. orEscherichia coli. More preferably, a polynucleotide transfected into thetransformed cell comprises the nucleotide base sequence of SEQ ID NOs:16 or 17. SEQ ID NOs: 16-17 and 13-14 set forth nucleotide and aminoacid sequences, respectively, for representative Salp15 polypeptides ofthe presently disclosed subject matter. Also provided are homologs orbiologically equivalent Salp15 polynucleotides and polypeptides found inother vertebrates besides tick species.

As mentioned above, in connection with expression embodiments to preparerecombinant Salp15 and peptides, it is provided that longer DNA segmentscan be used, with DNA segments encoding the entire Salp15 protein,biologically active domains or cleavage products thereof, being mostpreferred. However, it will be appreciated that the use of shorter DNAsegments to direct the expression of Salp15 peptides, epitopes or coreregions, such as can be used to generate anti-Salp15 antibodies, alsofalls within the scope of the presently disclosed subject matter.

DNA segments which encode peptide antigens from about 5 to about 50amino acids in length, or more preferably, from about 10 to about 30amino acids in length are contemplated to be particularly useful. DNAsegments encoding peptides will generally have a minimum coding lengthin the order of about 15 to about 150, or to about 90 nucleotides. DNAsegments encoding full-length proteins can have a minimum coding lengthon the order of about 400 or 500 nucleotides for a protein in accordancewith SEQ ID NO 1.

III.B. Peptide Modification Techniques and Derivatives

A Salp15 polypeptide or biologically functional equivalents thereof ofthe presently disclosed subject matter can be subject to variouschanges, substitutions, insertions, and deletions where such changesprovide for certain advantages in its use. Thus, the term “polypeptide”,“gene product” and “peptide” encompasses any of a variety of forms ofpeptide derivatives, that include amides, conjugates with proteins,cyclized peptides, polymerized peptides, conservatively substitutedvariants, analogs, fragments, peptoids, chemically modified peptides,and peptide mimetics. The modifications disclosed herein can also beapplied as desired and as appropriate to antibodies.

Additional residues can also be added at either terminus of a peptidefor the purpose of providing a “linker” by which the peptides of thepresently disclosed subject matter can be conveniently affixed to alabel or solid matrix, or carrier. Amino acid residue linkers areusually at least one residue and can be 40 or more residues, more often1 to 10 residues, but do alone not constitute radiation inducible targetligands. Typical amino acid residues used for linking are tyrosine,cysteine, lysine, glutamic and aspartic acid, or the like. In addition,a peptide can be modified by terminal-NH₂ acylation (e.g., acetylation,or thioglycolic acid amidation) or by terminal-carboxylamidation (e.g.,with ammonia, methylamine, and the like terminal modifications).Terminal modifications are useful, as is well known, to reducesusceptibility by proteinase digestion, and therefore serve to prolonghalf-life of the peptides in solutions, particularly biological fluidswhere proteases can be present.

Peptides of the presently disclosed subject matter can comprisenaturally occurring amino acids, synthetic amino acids, geneticallyencoded amino acids, non-genetically encoded amino acids, andcombinations thereof. Peptides can include both L-form and D-form aminoacids.

Representative non-genetically encoded amino acids include but are notlimited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionicacid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid);6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid;3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid;desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid;N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine;3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline;norvaline; norleucine; and ornithine.

Representative derivatized amino acids include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups can be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups canbe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine can be derivatized to form N-im-benzylhistidine.

III.B.1. Peptide Synthesis and Modification

Production of and modifications to the Salp15 proteins and peptidesdescribed herein can be carried out using techniques known in the art,including site directed mutagenesis and chemical synthesis.

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants; for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 30nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art as exemplified by publications (e.g., Adelman et al., 1983;Sambrook et al., 2001) and can be achieved in a variety of waysgenerally known to those of skill in the art.

Peptides of the presently disclosed subject matter, including peptoids,can also be chemically synthesized by any of the techniques that areknown to those skilled in the art of peptide synthesis. Syntheticchemistry techniques, such as a solid-phase Merrifield-type synthesis,can be used for reasons of purity, antigenic specificity, freedom fromundesired side products, ease of production, and the like. A summary ofrepresentative techniques can be found in Stewart & Young, 1969;Merrifield, 1969; Fields & Noble, 1990; and Bodanszky, 1993. Solid phasesynthesis techniques can be found in Andersson et al., 2000, and in U.S.Pat. Nos. 6,015,561; 6,015,881; 6,031,071; and 4,244,946. Peptidesynthesis in solution is described by Schröder & Lübke, 1965.Appropriate protective groups usable in such synthesis are described inthe above texts and in McOmie, 1973. In addition, peptides comprising aspecified amino acid sequence can be purchased from commercial sources(e.g., Biopeptide Co., LLC of San Diego, Calif., United States ofAmerica and PeptidoGenics of Livermore, Calif., United States ofAmerica).

III.B.2. Cyclic Peptides

Peptide cyclization is a useful modification because of the stablestructures formed by cyclization and in view of the biologicalactivities observed for such cyclic peptides as described herein. Anexemplary method for cyclizing peptides is described by Schneider &Eberle, 1993. Typically, tertbutoxycarbonyl protected peptide methylester is dissolved in methanol and sodium hydroxide solution are addedand the admixture is reacted at 20° C. to hydrolytically remove themethyl ester protecting group. After evaporating the solvent, thetertbutoxycarbonyl protected peptide is extracted with ethyl acetatefrom acidified aqueous solvent. The tertbutoxycarbonyl protecting groupis then removed under mildly acidic conditions in dioxane cosolvent. Theunprotected linear peptide with free amino and carboxyl termini soobtained is converted to its corresponding cyclic peptide by reacting adilute solution of the linear peptide, in a mixture of dichloromethaneand dimethylformamide, with dicyclohexylcarbodiimide in the presence of1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclicpeptide is then purified by chromatography.

III.B.3. Peptoids

The term “peptoid” as used herein refers to a peptide wherein one ormore of the peptide bonds are replaced by pseudopeptide bonds includingbut not limited to a carba bond (CH₂—CH₂), a depsi bond (CO—O), ahydroxyethylene bond (CHOH—CH₂), a ketomethylene bond (CO—CH₂), amethylene-oxy bond (CH₂—O), a reduced bond (CH₂—NH), a thiomethylenebond (CH₂—S), a thiopeptide bond (CS—NH), and an N-modified bond(—NRCO—). See e.g. Corringer et al., 1993; Garbay-Jaureguiberry et al.,1992; Tung et al., 1992; Urge et al., 1992; Pavone et al., 1993.

III.B.4. Peptide Mimetics

The term “peptide mimetic” as used herein refers to a ligand that mimicsthe biological activity of a reference peptide, by substantiallyduplicating the targeting activity of the reference peptide, but it isnot a peptide or peptoid. In one embodiment, a peptide mimetic is asmall molecule having a molecular weight of less than about 700 daltons.

A peptide mimetic can be designed by: (a) identifying the pharmacophoricgroups responsible for the targeting activity of a peptide; (b)determining the spatial arrangements of the pharmacophoric groups in theactive conformation of the peptide; and (c) selecting a pharmaceuticallyacceptable template upon which to mount the pharmacophoric groups in amanner that allows them to retain their spatial arrangement in theactive conformation of the peptide. For identification of pharmacophoricgroups responsible for targeting activity, mutant variants of thepeptide can be prepared and assayed for targeting activity.Alternatively or in addition, the three-dimensional structure of acomplex of the peptide and its target molecule can be examined forevidence of interactions, for example the fit of a peptide side chaininto a cleft of the target molecule, potential sites for hydrogenbonding, etc. The spatial arrangements of the pharmacophoric groups canbe determined by NMR spectroscopy or X-ray diffraction studies. Aninitial three-dimensional model can be refined by energy minimizationand molecular dynamics simulation. A template for modeling can beselected by reference to a template database and will typically allowthe mounting of 2-8 pharmacophores. A peptide mimetic is identifiedwherein addition of the pharmacophoric groups to the template maintainstheir spatial arrangement as in the peptide.

A peptide mimetic can also be identified by assigning a hashed bitmapstructural fingerprint to the peptide based on its chemical structure,and determining the similarity of that fingerprint to that of eachcompound in a broad chemical database. The fingerprints can bedetermined using fingerprinting software commercially distributed forthat purpose by Daylight Chemical Information Systems, Inc. (MissionViejo, Calif., U.S.A.) according to the vendor's instructions.Representative databases include but are not limited to SPREI'95(InfoChem GmbH of München, Germany), Index Chemicus (ISI ofPhiladelphia, Pa., U.S.A.), World Drug Index (Derwent of London, UnitedKingdom), TSCA93 (United States Environmental Protection Agency),MedChem (Biobyte of Claremont, Calif., U.S.A.), Maybridge OrganicChemical Catalog (Maybridge of Cornwall, England), Available ChemicalsDirectory (MDL Information Systems of San Leandro, Calif., U.S.A.),NCI96 (United States National Cancer Institute), Asinex Catalog ofOrganic Compounds (Asinex Ltd. of Moscow, Russia), and NP(InterBioScreen Ltd. of Moscow, Russia). A peptide mimetic of areference peptide is selected as comprising a fingerprint with asimilarity (Tanamoto coefficient) of at least 0.85 relative to thefingerprint of the reference peptide. Such peptide mimetics can betested for binding to a substrate molecule, such as for example the CD4co-receptor of T cells using the methods disclosed herein.

Additional techniques for the design and preparation of peptide mimeticscan be found in U.S. Pat. Nos. 5,811,392; 5,811,512; 5,578,629;5,817,879; 5,817,757; and 5,811,515.

III.B.5. Salts of Compositions

Any peptide or peptide mimetic of the presently disclosed subject mattercan be used in the form of a pharmaceutically acceptable salt. Suitableacids which are capable of the peptides with the peptides of thepresently disclosed subject matter include inorganic acids such astrifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid,perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoricacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of thepresently disclosed subject matter include inorganic bases such assodium hydroxide, ammonium hydroxide, potassium hydroxide and the like;and organic bases such as mono-di- and tri-alkyl and aryl amines (e.g.triethylamine, diisopropyl amine, methyl amine, dimethyl amine and thelike), and optionally substituted ethanolamines (e.g. ethanolamine,diethanolamine and the like).

IV. Introduction of Gene Products

In accordance with the present subject matter, where a Salp15 geneitself is employed to introduce a Salp15 gene product, a convenientmethod of introduction will be through the use of a recombinant vectorthat incorporates the desired gene, together with its associated controlsequences. The preparation of recombinant vectors is well known to thoseof skill in the art and described in many references, such as, forexample, Sambrook et al., 2001, incorporated herein in its entirety.

IV.A. Vector Construction

It is understood that the DNA coding sequences to be expressed, in thiscase those encoding the Salp15 gene products, are positioned in a vectoradjacent to and operatively linked to a promoter (i.e., under thecontrol of a promoter). It is understood in the art that to bring acoding sequence under the control of such a promoter, one generallypositions the 5′ end of the transcription initiation site of thetranscriptional reading frame of the gene product to be expressedbetween about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of)the chosen promoter.

One can also desire to incorporate into the transcriptional unit of thevector an appropriate polyadenylation site (e.g., 5″-AATAAA-3′), if onewas not contained within the original inserted DNA. Typically, thesepoly-A addition sites are placed about 30 to 2000 nucleotides“downstream” of the coding sequence at a position prior to transcriptiontermination.

While use of the control sequences of the specific gene will bepreferred, other control sequences can be employed, so long as they arecompatible with the genotype of the cell being treated. Thus, one canmention other useful promoters by way of example, including, e.g., anSV40 early promoter, a long terminal repeat promoter from retrovirus, anactin promoter, a heat shock promoter, a metallothionein promoter, andthe like.

As is known in the art, a promoter is a region of a DNA moleculetypically within about 100 nucleotide pairs upstream of (i.e., 5′ to)the point at which transcription begins (i.e., a transcription startsite). That region typically contains several types of DNA sequenceelements that are located in similar relative positions in differentgenes.

Another type of discrete transcription regulatory sequence element is anenhancer. An enhancer imposes specificity of time, location andexpression level on a particular coding region or gene. A major functionof an enhancer is to increase the level of transcription of a codingsequence in a cell that contains one or more transcription factors thatbind to that enhancer. An enhancer can function when located at variabledistances from transcription start sites so long as a promoter ispresent.

As used herein, the phrase “enhancer-promoter” means a composite unitthat contains both enhancer and promoter elements. An enhancer-promoteris operatively linked to a coding sequence that encodes at least onegene product. As used herein, the phrase “operatively linked” means thatan enhancer-promoter is connected to a coding sequence in such a waythat the transcription of that coding sequence is controlled andregulated by that enhancer-promoter. Techniques for operatively linkingan enhancer-promoter to a coding sequence are well known in the art; theprecise orientation and location relative to a coding sequence ofinterest is dependent, inter alia, upon the specific nature of theenhancer-promoter.

An enhancer-promoter used in a vector construct of the present subjectmatter can be any enhancer-promoter that drives expression in a cell tobe transfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

For introduction of a Salp15 gene, a vector construct that will deliverthe gene to cells of interest is desired. Viral vectors can be used.These vectors can optionally be a HSV-1, an adenovirus, a retrovirus,such as a Lentivirus, a vaccinia virus vector or an adeno-associatedvirus; these vectors have been successfully used to deliver desiredsequences to cells and tend to have a high infection efficiency.Suitable vector-Salp15 gene constructs are adapted for administration aspharmaceutically acceptable formulation, as described herein below.Viral promoters can also be of use in vectors of the present subjectmatter, and are known in the art.

Commonly used viral promoters for expression vectors are derived frompolyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40). Theearly and late promoters of SV40 virus are particularly useful becauseboth are obtained easily from the virus as a fragment that also containsthe SV40 viral origin of replication. Smaller or larger SV40 fragmentscan also be used, provided there is included the approximately 250 basepair sequence extending from the Hind III site toward the Bgl I sitelocated in the viral origin of replication. Further, it is alsopossible, and often desirable, to utilize promoter or control sequencesnormally associated with the desired gene sequence, provided suchcontrol sequences are compatible with the host cell systems.

The origin of replication can be provided either by construction of thevector to include an exogenous origin, such as can be derived from SV40or other viral source, or can be provided by the host cell chromosomalreplication mechanism. If the vector is integrated into the host cellchromosome, the latter is often sufficient.

As described herein, bacterial host cells can also be used to deliver aSalp15 to a cell or tissue of interest. See e.g., Motameni et al., 2004for representative methods for delivering Salp15 genes and vectors totarget tissues using a bacterial host cell. The Salp15 gene can beoperatively linked to a eukaryotic promoter in these vectors such thatthe gene is not transcribed prior to delivery to the cell or tissue ofinterest.

Where a Salp15 gene itself is employed it will be most convenient tosimply use a wild type Salp15 gene directly. However, it is providedthat certain regions of a Salp15 gene can be employed exclusivelywithout employing an entire wild type Salp15 gene, including fragments,and particularly fragments encoding the C-terminus of Salp15.Optionally, the smallest region needed to modulate biological activityso that one is not introducing unnecessary DNA into cells that receive aSalp15 gene construct can be employed. The ability of these regions tomodulate biological activity can be determined by the assays reported inthe EXAMPLES.

V. Methods Employing the Presently Disclosed Compositions

The presently disclosed subject matter provides isolated and purifiedbiologically active Salp15 polypeptides and nucleic acid moleculesencoding same. As disclosed herein, the Salp15 polypeptides are capableof specifically binding to CD4⁺ T cells and modulating Class IIMHC-mediated T-lymphocyte activation via the T cell receptor activationpathway of the CD4⁺ T cells. The Salp15 polypeptides disclosed hereincan modulate T cell activation via the specific binding to CD4 receptormolecules expressed on the surface of T cells. More particularly, theSalp15 polypeptides can bind the extracellular outer two domains (D1-D2)region of the CD4 receptor and thereby prevent propagation of theTCR-mediated activation signal, inhibiting activation of the T cell. Thepresently disclosed subject matter provides methods of employing theseunique properties of Salp15 polypeptides, as discussed herein below.

V.A. Methods of Modulating T Cell Activation

The presently disclosed subject matter provides in some embodiments amethod of modulating activation of a CD4⁺ T cell due to T cellreceptor-mediated signaling, comprising contacting the T cell with aSalp15 polypeptide as disclosed herein, where activation of the T cellis modulated. In some embodiments, T cell activation is inhibited bycontacting the T cell with the Salp15 polypeptide. Thus, activation ofthe T cell (or a population of T cells overall) is decreased orsubstantially completely prevented. By inhibiting activation of the Tcell, the T cell is unable to proliferate and differentiate, therebydampening the immune response that otherwise would have been produced bythe activated T cell or population of T cells.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 16and 17; a polypeptide encoded by a nucleic acid having at least about90% or greater sequence identity to a DNA sequence as set forth in anyof SEQ ID NOs: 16 and 17; a polypeptide having an amino acid sequence ofany of SEQ ID NOs: 13 and 14, or a biologically functional equivalentthereof; a polypeptide which is immunologically cross-reactive withantibodies which are immunologically reactive with a polypeptide havingan amino acid sequence of any of SEQ ID NOs: 13 and 14; or a fragment ofone of these Salp15 polypeptides.

In some embodiments, the Salp15 polypeptide is contacted with a T cellcultured in vitro. In other embodiments, the T cell is found within asubject and the Salp15 is contacted with the T cell by administering theSalp15 polypeptide to the subject. Therapeutic methods of administrationare described in detail elsewhere herein.

V.B. Methods of Treating Disorders in Subjects

As discussed hereinabove and in the EXAMPLES, Salp15 polypeptides of thepresently disclosed subject matter are useful to modulate CD4+ T cellactivation. The Salp15 polypeptides bind CD4 receptor and inhibitactivation of the T cell via the TCR-mediated signaling pathway,resulting in inhibition of IL-2 production and/or CD25 production (theIL-2 receptor). IL-2 mediated signaling is an important step in naïve Tcell activation, and ultimately proliferation and differentiation. Thus,Salp15 polypeptides of the presently disclosed subject matter caninhibit inflammatory responses due at least in part to naïve CD4⁺ T cellactivation and therefore are useful for the treatment of disorderscharacterized by the activation of T cells, including but not limited toautoimmune disorders and tissue and organ transplant rejection.

V.B.1. Methods of Treating T Cell Response-Related Conditions

As such, the presently disclosed subject matter provides methods oftreating a subject suffering from or at risk of suffering from acondition characterized by a CD4⁺ T cell response, comprisingadministering to the subject an effective amount of a Salp15 polypeptideas disclosed herein.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 16and 17; a polypeptide encoded by a nucleic acid having at least about90% or greater sequence identity to a DNA sequence as set forth in anyof SEQ ID NOs: 16 and 17; a polypeptide having an amino acid sequence ofany of SEQ ID NOs: 13 and 14, or a biologically functional equivalentthereof; a polypeptide which is immunologically cross-reactive withantibodies which are immunologically reactive with a polypeptide havingan amino acid sequence of any of SEQ ID NOs: 13 and 14; or a fragment ofone of these Salp15 polypeptides.

In some embodiments the condition treated with the Salp15 polypeptiderelates to inhibiting (reducing the risk of) rejection of transplantedtissues and/or organs. In these embodiments, the Salp15 polypeptide canbe administered prior to, in conjunction with, and/or aftertransplantation of the tissue or organ. In some embodiments, thecondition is an autoimmune disorder. Exemplary autoimmune disordersinclude but are not limited to lupus, rheumatoid arthritis, type 1diabetes, multiple sclerosis, rheumatic fever, and Hashimoto's disease.

Effective dosages and formulations are chosen according to a number offactors, including but not limited to the particular subject, thecondition treated and the severity of the condition, for example.Guidance regarding subjects treated, dosages and formulations isprovided in detail elsewhere herein.

V.B.2. Methods of Treating Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the centralnervous system (CNS). Despite intensive investigation, the mechanisms ofdisease pathogenesis remain unclear, and curative therapies areunavailable for MS. Genetic predisposition, epidemiologic factors, andautoimmunity are all thought to be involved in the pathogenesis of MS(Vanderlugt et al., 2000). Self-reactive T cell activation and innateimmune cell activation are thought to be major eliciting factors for theappearance of the symptomatology associated with the damage of themyelin sheath (Kuchroo et al., 2002). The inflammation associated withthe disease is believed to be caused by the activation of CD4⁺ T cellswith a Th1 phenotype that produce IFN(, activate innate immune cells andinduce the production of complement-fixing antibody isotypes (Tran etal., 2002).

The study of MS has advanced dramatically with the use of differentrodent models, including rats and mice. These studies have allowed abetter understanding of how the rodent MS-like syndrome can beregulated, leading to therapeutic modalities which have been applied inclinical studies of MS patients. Some strains of mice develop ademyelinating inflammatory disease of the central nervous system knownas Relapsing-remitting Experimental Autoimmune Encephalomyelitis (R-EAE)that mimics MS (McRae et al., 1992). R-EAE can be induced in a number ofmouse strains (SJL, (SWR×SJL)F₁). These mice are especially usefulbecause the specificity of the eliciting antigen has been characterizedto the peptide level and the hierarchy of epitope spreading has beendetermined (Lehmann et al., 1992; McRae et al., 1995; Yu et al., 1996).The animals, when immunized with a specific peptide from the myelinproteolipid protein (PLP) or Myelin Basic Protein (MBP) develop theMS-like syndrome R-EAE. The disease course in mice is similar to thatfound in humans: an initial bout of disease is followed by the remissionof the symptoms (Goverman & Brabb, 1996).

Evidence exists that points to reactivation of memory T cells and/orepitope spreading (i.e., neoautoreactivity) to explain the chronicityand the relapsing-remitting clinical course often associated with thedisease (Vanderlugt et al., 2000; McRae et al., 1995; Lehrnann et al.,1993). The animals present with relapsing episodes that are at leastpartially due to epitope spreading, which gives rise to the activationof new CD4⁺ T cells recognizing other CNS peptides. These includepeptides present in PLP (intramolecular epitope spreading) or otherproteins (MBP, MOG; intermolecular epitope spreading) (McRae et al.,1995). Therefore, control of these relapsing episodes could provide away to evade progression of disease. Experiments carried out in themurine model have indicated that neoreactive CD4⁺ T cells are able toinduce relapsing episodes, since 1) CD4⁺ T cells specific torelapse-associated epitopes can transfer the disease to naïve animals(Vanderlugt et al., 2000; McRae et al., 1995; Yu et al., 1996), 2)induction of peptide-specific tolerance to relapsing-associated epitopesduring remission from acute disease prevents the progression of thedisease (Vanderlugt et al., 2000), and 3) manipulation of costimulatorypathways required for T cell activation also modulates diseaseprogression (Vanderlugt et al., 2000; Karandikar et al., 2000). Thus, itis likely that the inhibition of T cell activation in response torelapsing-associated epitopes during remission of the acute phase of thedisease can reduce or even prevent the appearance of relapsing episodes.

Peptide specific disease therapy is difficult due to the diversity ofpeptides that can trigger relapsing episodes in humans. Alternativeapproaches include the inhibition of T cell activation using blockingantibodies to costimulatory molecules. The presently disclosed subjectmatter provides methods for administering Salp15 polypeptides disclosedherein as a treatment of patients with MS.

In some embodiments, the presently disclosed subject matter furtherprovides a method of treating multiple sclerosis in a subject,comprising administering to the subject an effective amount of a Salp15polypeptide as disclosed herein, or a biologically active fragmentthereof, having immunosuppressive activity to the subject.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in SEQ ID NO: 1; apolypeptide encoded by a nucleic acid having at least about 90% orgreater sequence identity to a DNA sequence as set forth in SEQ ID NO:1; a polypeptide encoded by a nucleic acid capable of hybridizing understringent conditions to a nucleic acid comprising a sequence or thecomplement of a sequence as set forth in SEQ ID NO: 1; a polypeptidehaving an amino acid sequence of SEQ ID NO: 2, or a biologicallyfunctional equivalent thereof; a polypeptide which is immunologicallycross-reactive with antibodies which are immunologically reactive with apolypeptide having an amino acid sequence of SEQ ID NO: 2; or a fragmentof one of these Salp15 polypeptides, including but not limited topolypeptides having an amino acid sequence of SEQ ID NOs: 13 or 14, or abiologically functional equivalent thereof.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 16and 17; a polypeptide encoded by a nucleic acid having at least about90% or greater sequence identity to a DNA sequence as set forth in anyof SEQ ID NOs: 16 and 17; a polypeptide having an amino acid sequence ofany of SEQ ID NOs: 13 and 14, or a biologically functional equivalentthereof; a polypeptide which is immunologically cross-reactive withantibodies which are immunologically reactive with a polypeptide havingan amino acid sequence of any of SEQ ID NOs: 13 and 14; or a fragment ofone of these Salp15 polypeptides.

V.C. Methods of Inhibiting Infection of T Cells by HIV

Human immunodeficiency virus (HIV) is the etiologic agent of acquiredimmunodeficiency syndrome (AIDS). HIV is a human retrovirus of theLentivirus group. The four recognized human retroviruses belong to twodistinct groups: the human T lymphotropic (or leukemia) retroviruses,HTLV-1 and HTLV-2, and the human immunodeficiency viruses, HIV-1 andHIV-2. The former are transforming viruses whereas the latter arecytopathic viruses. As used herein, “HIV” refers to HIV-1 and HIV-2, andvariant strains thereof.

The common denominator of AIDS is a profound immunosuppression,predominantly of cell-mediated immunity. This immune suppression leadsto a variety of opportunistic diseases, particularly certain infectionsand neoplasms. The main cause of the immune defect in AIDS has beenidentified as a quantitative and qualitative deficiency in the CD4+ Tcells, which has been demonstrated to be the cellular receptor for HIV.Dalgleish et al., 1984. Although the T4 cell is the major cell typeinfected with HIV, essentially any human cell that expresses the CD4molecule on its surface is capable of binding to and being infected withHIV.

HIV binds specifically and with high affinity, via a stretch of aminoacids in the viral envelope protein Env (gp120), to a portion of the D1region of the CD4 receptor located near its N-terminus. Followingbinding, the virus fuses with the target cell membrane and isinternalized. Once internalized it uses the enzyme reverse transcriptaseto transcribe its genomic RNA to DNA, which is integrated into thecellular DNA where it exists for the life of the cell as a “provirus.”

The provirus may remain latent or be activated to transcribe mRNA andgenomic RNA, leading to protein synthesis, assembly, new virionformation, and budding of virus from the cell surface. Although theprecise mechanism by which the virus induces cell death has not beenestablished, it is believed that the major mechanism is massive viralbudding from the cell surface, leading to disruption of the plasmamembrane and resulting osmotic disequilibrium.

During the course of the infection, the host organism developsantibodies against viral proteins, including the major envelopeglycoproteins gp120 and gp41. Despite this humoral immunity, the diseaseprogresses, resulting in a lethal immunosuppression characterized bymultiple opportunistic infections, parasitemia, dementia, and death. Thefailure of the host anti-viral antibodies to arrest the progression ofthe disease represents one of the most vexing and alarming aspects ofthe infection, and augurs poorly for vaccination efforts based uponconventional approaches.

Evidence that the CD4-gp120 binding is responsible for viral infectionof cells bearing the CD4 antigen (along with gp120 binding to achemokine co-receptor) includes the finding that a specific complex isformed between gp120 and CD4 (McDougal et al., 1986). Otherinvestigators have shown that cell lines, which were non-infective forHIV, were converted to infectable cell lines following transfection andexpression of the human CD4 cDNA gene. Maddon et al., 1986.

Methods have previously been proposed for blocking HIV infection of Tcells using molecules that can specifically block the binding of HIVgp120 to T cell CD4, thereby preventing infection of the T cells by HIV.For example, Capon et al. proposed the use of fusion polypeptidescomprising the soluble four domains of CD4 (D1-D4) and an immunoglobulinFc region, suggesting the fusion peptide would bind HIV gp120 and blockgp120 interaction with CD4 on T cells. See Capon et al., 1989,incorporated herein by reference in its entirety. Arenzanz-Seisdedos etal. proposed utilizing a fragment of the RANTES chemokine having bindingspecificity for the CCR5 receptor, a co-receptor (along with CD4)utilized by HIV for infection of T cells. It was proposed the RANTESfragment could possibly inhibit infection by blocking gp120 interactionwith CCR5. See Arenzanz-Seisdedos et al., 1996, incorporated herein byreference in its entirety.

The presently disclosed subject matter provides methods of blocking HIVinfection by blocking effective gp120 binding to CD4. As disclosedherein, Salp15 binds with specificity at the D1-D2 domains of CD4. Asdemonstrated in the EXAMPLES, specific binding of Salp15 to CD4 caneffectively block binding of gp120 to CD4. Thus, Salp15 polypeptidesdisclosed herein can be utilized to inhibit HIV infection of T cells,including in vivo, by blocking gp120 binding of CD4. One of skill in theart will appreciate that in some circumstances, it will be desirable toutilize Salp15 polypeptides, fragments, or mimetics thereof that onlyblock binding of gp120 to CD4 and do not further cause modification toCD4 structure such that T cell activation is inhibited resulting inimmunomodulation. Such peptides could be particularly preferred for usein subjects whose immune systems are previously suppressed, due to forexample previous infection by HIV. Therefore, Salp15 polypeptidesexhibiting these qualities are encompassed by the presently disclosedsubject matter as well.

In some embodiments, the presently disclosed subject matter provides amethod of inhibiting infection of a T cell by a human immunodeficiencyvirus (HIV), comprising contacting a CD4 receptor expressed by the Tcell with a Salp15 polypeptide, or a biologically active fragmentthereof having binding specificity for the CD4 receptor, wherebycontacting the Salp15 polypeptide with the CD4 receptor inhibits the HIVfrom infecting the T cell.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in SEQ ID NO: 1; apolypeptide encoded by a nucleic acid having at least about 90% orgreater sequence identity to a DNA sequence as set forth in SEQ ID NO:1; a polypeptide encoded by a nucleic acid capable of hybridizing understringent conditions to a nucleic acid comprising a sequence or thecomplement of a sequence as set forth in SEQ ID NO: 1; a polypeptidehaving an amino acid sequence of SEQ ID NO: 2, or a biologicallyfunctional equivalent thereof; a polypeptide which is immunologicallycross-reactive with antibodies which are immunologically reactive with apolypeptide having an amino acid sequence of SEQ ID NO: 2; or a fragmentof one of these Salp15 polypeptides, including but not limited topolypeptides having an amino acid sequence of SEQ ID NOs: 13 or 14, or abiologically functional equivalent thereof.

In some embodiments, the Salp15 polypeptide comprises a polypeptideencoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 16and 17; a polypeptide encoded by a nucleic acid having at least about90% or greater sequence identity to a DNA sequence as set forth in anyof SEQ ID NOs: 16 and 17; a polypeptide having an amino acid sequence ofany of SEQ ID NOs: 13 and 14, or a biologically functional equivalentthereof; a polypeptide which is immunologically cross-reactive withantibodies which are immunologically reactive with a polypeptide havingan amino acid sequence of any of SEQ ID NOs: 13 and 14; or a fragment ofone of these Salp15 polypeptides.

In some embodiments, the Salp15 polypeptide is contacted with a T cellcultured in vitro. In other embodiments, the T cell is found within asubject and the Salp15 is contacted with the T cell by administering theSalp15 polypeptide to the subject. Methods of administration aredescribed in detail elsewhere herein.

V.D. Subjects

Further with respect to the therapeutic methods of the presentlydisclosed subject matter, a preferred subject is a vertebrate subject. Apreferred vertebrate is warm-blooded; a preferred warm-bloodedvertebrate is a mammal. A preferred mammal is most preferably a human.As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently disclosed subject matter.

As such, the presently disclosed subject matter provides for thetreatment of mammals such as humans, as well as those mammals ofimportance due to being endangered, such as Siberian tigers; of economicimportance, such as animals raised on farms for consumption by humans;and/or animals of social importance to humans, such as animals kept aspets or in zoos. Examples of such animals include but are not limitedto: carnivores such as cats and dogs; swine, including pigs, hogs, andwild boars; ruminants and/or ungulates such as cattle, oxen, sheep,giraffes, deer, goats, bison, and camels; and horses. Also provided isthe treatment of birds, including the treatment of those kinds of birdsthat are endangered and/or kept in zoos, as well as fowl, and moreparticularly domesticated fowl, i.e., poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomic importance to humans. Thus, also provided is the treatment oflivestock, including, but not limited to, domesticated swine, ruminants,ungulates, horses (including race horses), poultry, and the like.

V.E. Formulations

A composition as described herein preferably comprises a compositionthat includes a carrier. In some embodiments, particularly with regardto the therapeutic methods, the carrier is a pharmaceutically acceptablecarrier in mammals, e.g. humans. Suitable formulations include aqueousand non-aqueous sterile injection solutions that can containantioxidants, buffers, bacteriostats, bactericidal antibiotics andsolutes that render the formulation isotonic with the bodily fluids ofthe intended recipient; and aqueous and non-aqueous sterile suspensions,which can include suspending agents and thickening agents. Thecomposition can be formulated according to the mode of administration,which can include, but is not limited to systemic administration,parenteral administration (including intravascular, intramuscular, andintraarterial administration), oral delivery, buccal delivery,subcutaneous administration, inhalation, intratracheal installation,surgical implantation, transdermal delivery, local injection, andhyper-velocity injection/bombardment or combinations thereof ofadministration modes.

The compositions used in the methods can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets can be coated by methods known in the art. For example, a Salp15polypeptide, including biologically active fragments and modifiedpolypeptides, and polypeptide mimetics, can be formulated in combinationwith hydrochlorothiazide, and as a pH stabilized core having an entericor delayed release coating which protects the active agents untilreaching desired regions of the gastrointestinal tract.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compounds can also be formulated as a preparation for implantationor injection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (e.g., as a sparingly soluble salt).

The compounds can also be formulated in rectal compositions (e.g.,suppositories or retention enemas containing conventional suppositorybases such as cocoa butter or other glycerides), creams or lotions, ortransdermal patches.

V.F. Doses

The term “effective amount” is used herein to refer to an amount of acomposition (e.g., a composition comprising a Salp15 polypeptide)sufficient to produce a measurable biological response (e.g., ameasurable inhibition in T cell activation). Actual dosage levels ofactive ingredients in a composition of the presently disclosed subjectmatter can be varied so as to administer an amount of the activecompound(s) that is effective to achieve the desired response for aparticular subject and/or application. The selected dosage level willdepend upon a variety of factors including the activity of thecomposition, formulation, the route of administration, combination withother drugs or treatments, severity of the condition being treated, andthe physical condition and prior medical history of the subject beingtreated. Preferably, a minimal dose is administered, and dose isescalated in the absence of dose-limiting toxicity to a minimallyeffective amount. Determination and adjustment of an effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine.

For administration of a composition as disclosed herein, conventionalmethods of extrapolating human dosage based on doses administered to amurine animal model can be carried out using the conversion factor forconverting the mouse dosage to human dosage: Dose Human per kg=DoseMouse per kg×12 (Freireich et al., 1966). Drug doses can also be givenin milligrams per square meter of body surface area because this methodrather than body weight achieves a good correlation to certain metabolicand excretionary functions. Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich et al., 1966.Briefly, to express a mg/kg dose in any given species as the equivalentmg/sq m dose, multiply the dose by the appropriate km factor. In anadult human, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/m².

For oral administration, a satisfactory result can be obtained employingthe Salp15 polypeptide in an amount ranging from about 0.01 mg/kg toabout 100 mg/kg and preferably from about 0.1 mg/kg to about 30 mg/kg. Apreferred oral dosage form, such as tablets or capsules, will containthe Salp15 polypeptide in an amount ranging from about 0.1 to about 500mg, preferably from about 2 to about 50 mg, and more preferably fromabout 10 to about 25 mg.

For parenteral administration, the Salp15 polypeptide can be employed inan amount ranging from about 0.005 mg/kg to about 100 mg/kg, preferablyabout 10 to 50 or 10 to 70 mg/kg, and more preferably from about 10mg/kg to about 30 mg/kg.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., 1997; Goodman et al., 1996; Ebadi, 1998;Katzung, 2001; Remington et al., 1975; Speight et al., 1997; and Duch etal., 1998.

V.G. Routes of Administration

Suitable methods for administering to a subject a Salp15 polypeptide inaccordance with the methods of the presently disclosed subject matterinclude but are not limited to systemic administration, parenteraladministration (including intravascular, intramuscular, andintraarterial administration), oral delivery, buccal delivery,subcutaneous administration, inhalation, intratracheal installation,surgical implantation, transdermal delivery, local injection, andhyper-velocity injection/bombardment. Where applicable, continuousinfusion can enhance drug accumulation at a target site (see e.g., U.S.Pat. No. 6,180,082).

The particular mode of administration used in accordance with themethods of the present subject matter depends on various factors,including but not limited to the vector and/or carrier employed, theseverity of the condition to be treated, and mechanisms for metabolismor removal of the drug following administration.

V.H. Screening Methods

As discussed herein, Salp15 can bind with specificity to anextracellular region of the CD4 co-receptor, which results in aninability of the CD4 receptor to participate effectively in theTCR-mediated signaling pathway. As a result, the T cell expressing thebound CD4 will be inhibited in whole or part from becoming activated bythe TCR-mediated signal, and therefore will not undergo theproliferation or differentiation process, and instead remainingquiescent. Further, as discussed herein, the presently disclosed subjectmatter provides compositions with the same biological activity asSalp15, which is based on an ability to bind also with specificity theCD4 receptor. Methods of screening for compounds with the desiredactivity are thus also provided.

A method of screening candidate substances for an ability to modulateactivation of a CD4⁺ T cell is provided in accordance with the presentlydisclosed subject matter. In some embodiments, the method comprises (a)establishing a test sample comprising a CD4 receptor polypeptide and aligand for the CD4 receptor polypeptide, wherein the ligand is a Salp15polypeptide; (b) administering a candidate substance or a samplesuspected of containing a candidate substance to the test sample; and(c) measuring an effect of the candidate substance on binding of the CD4receptor polypeptide and the ligand in the test sample. In someembodiments, the measuring can comprise determining the ability of thecandidate substance to modulate activation of a CD4⁺ T cell resultingfrom T cell receptor-mediated signaling. Further, in some embodiments,the measuring can comprise determining whether or not the candidatesubstance can block binding of the CD4 receptor polypeptide and theligand.

The test sample can further comprise an indicator. The term “indicator”is meant to refer to a chemical species or compound that is readilydetectable using a standard detection technique, such as dark versuslight detection, fluorescence or chemiluminescence spectrophotometry,scintillation spectroscopy, chromatography, liquid chromatography/massspectroscopy (LC/MS), colorimetry, and the like. Representativeindicator compounds thus include, but are not limited to, fluorogenic orfluorescent compounds, chemiluminescent compounds, colorimetriccompounds, UV/VIS absorbing compounds, radionucleotides and combinationsthereof. In a preferred embodiment, the ligand further comprises anindicator.

The ability of the candidate substance to modulate activation of a CD4⁺T cell can determined in any suitable manner. For example, the abilityof the candidate substance to modulate activation of a CD4⁺ T cell candetermined by: (i) detecting a signal produced by the indicator upon aneffect of the candidate substance on binding of the CD4 receptorpolypeptide and the ligand; and (ii) identifying the candidate substanceas a modulator of activation of a CD4⁺ T cell based upon an amount ofsignal produced as compared to a control sample.

In some embodiments, a fluorescence based screening methodology isutilized to identify compositions that can bind with specificity at aregion of the CD4 receptor such that the composition competitivelyinhibits the ligand Salp15 from binding the CD4 receptor. The method isreadily amenable to both robotic and very high throughput systems.

Thus, in one embodiment, a screening method of the present subjectmatter pertains to a method for a identifying a candidate substance foran ability to modulate activation of a CD4⁺ T cell due to T cellreceptor-mediated signaling. The method comprises establishing a testsample comprising a CD4 receptor and a candidate substance,administering to the test sample a Salp15 peptide (a ligand for CD4)comprising an indicator, incubating the sample for a sufficient time toallow interaction of the ligand with the CD4 receptor; and detecting asignal produced by the indicator; and identifying the candidatesubstance as having an ability to modulate activation of a CD4⁺ T celldue to T cell receptor-mediated signaling based upon an amount of signalproduced by the indicator as compared to a control sample, which did notcontain the candidate substance. In the presence of a candidatesubstance capable of binding CD4 at the same region as Salp15, thecandidate substance will compete for binding of CD4 with Salp15. Thegreater the affinity of the candidate substance for CD4 at the regionwhere Salp15 binds, the lower the amount of signal produced and the morepromising the candidate substance as a modulator of T cell activation.

In some embodiments, the candidate substance is a polypeptide, and insome embodiments, the polypeptide is an antibody or functionalequivalent fragment thereof. Functional fragments of antibodies aredescribed in detail herein. In some embodiments, a nucleic acid moleculeencoding the candidate polypeptide is isolated and purified.Alternatively, in some embodiments, the candidate substance is a smallmolecule, such as a peptide mimetic of Salp15. Peptide mimetics aredescribed in detail elsewhere herein.

In another embodiment of the screening method of the presently disclosedsubject matter, a Salp15 polypeptide or active fragment thereof, and aCD4 receptor can be used for screening libraries of compounds in any ofa variety of high throughput drug screening techniques. The componentsemployed in such screening may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Forexample, in some embodiments, the CD4 receptor or an extracellularfragment thereof is immobilized to a solid support and the Salp15polypeptides and candidate substances are allowed to compete for bindingto the immobilized CD4 receptor. The solid support can then be easilywashed to remove unbound substances. The formation of binding complexes,between the Salp15 polypeptide or candidate substance with the CD4receptor, can then be measured as described herein.

EXAMPLES

The following EXAMPLES provide illustrative embodiments. Certain aspectsof the following EXAMPLES are disclosed in terms of techniques andprocedures found or contemplated by the present inventors to work wellin the practice of the embodiments. In light of the present disclosureand the general level of skill in the art, those of skill willappreciate that the following EXAMPLES are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently claimedsubject matter.

Materials and Methods for Examples 1-5

Cell purification and Activation. Naïve CD4+ T cells from C3H/HeN,Balb/c, and B10.BR mice were purified as previously described (Anguitaet al., 2002). CD4+ T cells were preincubated in Click's modifiedBruff's Medium (Sigma Chemical Co., St. Louis, Mo., U.S.A.) for 10minutes at 37° C. with and without Salp15 (50 μg/mL) and stimulated for20 min or the indicated times at 37° C. with 10 μg/mL of anti-CD3ε, 1μg/mL of anti-CD28, and 10 μg/mL of anti-armenian and syrian hamster IgG(BD Biosciences Pharmingen, San Jose, Calif., U.S.A.). Jurkat (E6-1)cells were stimulated using 5 μg/mL of OKT3 mAb (eBioscience, San Diego,Calif., U.S.A.) and 1 μg/mL of anti-human CD28 cross linked with 5 μg/mLanti-mouse IgG for the indicated times.

Antibodies. The anti-p-LAT (Tyr191), anti-pSrc family (Tyr416),anti-LCK, anti-Vav1, anti-pZap70 (Tyr319), anti-Zap-70, p44/42, p44/42(phopho-Tyr204/Thr202), anti-pSTAT1 and anti-STAT1 were purchased formCell Signaling Technologies (Beverly, Mass., U.S.A.). Anti-pTyr (pY99),anti-pPLCγ1 (Tyr783), anti-PLCγ1, polyclonal anti-CD4, MT310, anti-CD3,anti-CD28, anti-TCRβ and normal rabbit IgG were purchased from SantaCruz Biotechnology (Santa Cruz, Calif., U.S.A.). The polyclonalanti-His-HRP antibody was purchased from Novus Biologicals (Littleton,Colo., U.S.A.). OKT4 was purchased from eBioscience.

Confocal Microscopy. For the visualization of lipid rafts,phospho-tyrosine, and cytoskeleton reorganization, CD4⁺, CD8⁺ or Jurkatcells were cytospun for 10 minutes at 1000 RPM onto positively chargedmicroslides (Fisher Scientific, Hampton, N.H., U.S.A.). The cells werefixed with 3.7% paraformaldehyde (Sigma Chemical Co.) for 15 minutes andstained with CTB₅₉₄ (Molecular Probes, Eugene, Oreg., U.S.A.),AlexaFluor₄₈₈ labeled phalloidin (Molecular Probes) or (1:50)biotin-conjugated anti-p-Tyr (PY99) followed byStreptavidin-AlexaFluor₅₉₄ (1:100, Molecular Probes). For p-LAT andp-PLCγ1 staining, fixed cells were treated with PBS containing 0.1%Triton-X 100 and 100 mM glycine for 15 min at room temperature andstained using rabbit p-LAT (Tyr¹⁹¹) or rabbit anti-p-PLCγ1 (Tyr⁷⁸³)probed with anti-rabbit IgG₄₈₈ (Molecular Probes).

For receptor capping studies, mouse primary B and CD11b⁺ cells werepurified by positive selection using biotin anti-mouse CD45R/B220 cloneRA3-6B2 and biotin anti-mouse CD11b clone M1/70 (BD BiosciencesPharmingen) respectively, followed by incubation with magneticmicrobeads. The cells were analyzed for receptor capping using 25:g/mLanti-mouse IgM and 25 μg/mL anti-mouse CD16/CD32 respectively,cross-linked with 25 μg/mL of anti-rat IgG594 for 10 min in the presenceor absence of 50 μg/mL of Salp15. CD4⁺ and CD8⁺ T cells were purified bynegative selection and incubated with 10 μg/mL anti-CD3 and 1 μg/mLanti-CD28 cross-linked with 10 μg/mL of anti-hamster IgG₄₈₈ for 10 minin the presence or absence of 50 μg/mL of Salp15.

For colocalization experiments, Jurkat and CD4⁺ T cells were cytospun asdescribed herein. HeLa and HeLa-CD4 cells were grown on chamber slides.The cells were fixed and stained with anti-CD4 and Salp15 as follows.Purified Salp15 was labeled using Alexa-488 with the Protein LabelingKit (Molecular Probes) according to the manufacturer's instructions.Fixed cells were blocked with 5% normal serum and anti-CD16/CD32 (1:100)in PBS, incubated with 1 μg of monoclonal biotinylated anti-CD4 (L3T4)(BD Biosciences Pharmingen) followed by streptavidin₅₉₄ staining. Thecells were then stained with 20 μg/mL of Salp15₄₈₈. For competitionassays, cells were first blocked with 5% normal serum, incubated witheither polyclonal anti-CD4 antibody, MT310, OKT4, normal rabbit IgG,Salp15 (0.5 mg/mL) or lysozyme (0.5 mg/mL) followed by staining withSalp15₄₈₈. All samples were mounted in a GVA Mount (Zymed, SanFrancisco, Calif., U.S.A.) and visualized with an Olympus Confocalmicroscope equipped with Fluoview 3.0 software.

Western blotting and co-immunoprecipitation. For tyrosinephosphorylation western analysis, cells were lysed in 60 mM Tris (pH7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM ∃-glycerolphosphate, 2.5 mM sodium pyrophosphate, 25 mM NaF, 2 mM sodiumorthovanadate, 1% Triton-X 100, 0.25 sodium deoxycholate, 1 mM PMSF andprotease inhibitors for 15 min on ice, sonicated 4×5 sec and incubatedat 37° C. for 10 min. The lysate was cleared by centrifugation andboiled in SDS-sample buffer. The samples were subjected to SDS-PAGE,transferred to a nitrocellulose membrane and immunoblotted withanti-pTyr, anti-pPLCγ1 and anti-Vav1, anti-pSrc family (Tyr⁴¹⁶) tomeasure p-Lck³⁹⁴ (Alonso et al., 2004), anti-Lck, anti-pZap70 (Tyr³¹⁹),anti-Zap70, p44/42 and p44/42 (phopho-Tyr²⁰⁴/Thr²⁰²). Vav1 wasimmunoprecipitated using anti-Vav1 and protein A-agarose beadsovernight, subjected to SDS-PAGE followed by immunoblotting withanti-pTyr or anti-Vav1. To examine the effect of Salp15 on T cellstimulation with INFγ, CD4⁺ T cells were preincubated with Salp15 at 50μg/mL followed by activation with 20 ng/mL of mouse recombinant INFγ (R& D Systems, Minneapolis, Minn., U.S.A.) for 30 min at 37° C. The cellswere lysed as above and subjected to SDS-PAGE and immunoblotting withanti-pSTAT1 and anti-STAT1.

For co-immunoprecipitation, 1-2×10⁷ Jurkat, HeLa or HeLa-CD4 cells werelysed in 20 mM HEPES pH 7.4 containing 150 mM NaCl, 1% Triton-X 100,0.25% sodium deoxycholate, 25 mM NaF, 2 mM Na₃VO₄, 1 mM PMSF andprotease inhibitors for 15 min on ice. One mg of protein equivalent ofthe cell lysate was mixed with 50 μg/mL of 6× histidine-tagged-Salp15 or6×His-tagged Salp13 fused to thioredoxin (29 kD) as a control protein,(Anguita et al., 2002, Das et al., 2001) and immunoprecipitated using ananti-poly-histidine antibody conjugated to agarose, anti-CD4, anti-CD3,anti-CD28 or normal rabbit IgG overnight at 4° C. The immunoprecipitatewas run on SDS-PAGE followed by immunoblotting with anti-poly His-HRP,anti-CD4, anti-CD3, anti-CD28 or anti-TCRβ.

Quantification of immunoblots was performed using the SCION IMAGE™software package, Scion Corporation (Frederick, Md., U.S.A.).

Flow cytometry. Actin polymerization assay was performed as describedearlier (Krishnan et al., 2004). Briefly, 0.5×10⁶ CD4+ T cells werefixed with 3.7% paraformaldehyde for 8 min. The fixed cells were washedwith PBS twice and permeabilized and stained with staining buffercontaining 0.1% Triton X-100, 0.2 μM phalloidin488, 5% BSA and 0.2 mg/mLAPC-conjugated L3T4 monoclonal antibody (BD Biosciences Pharmingen) for40 min. The cells were washed, resuspended in PBS and analyzed forF-actin content on a FACSCalibur with CellQuest software (BecktonDickinson, Mountain View, Calif., U.S.A.). For the Salp15 bindingassays, the suspended cells were first blocked with PBS containing 5%BSA and 0.1% sodium azide for 10 min, washed with PBS containing 1% BSAfollowed by staining with Salp15 binding solution containing 15 μg/106cells of Salp15488, 5% BSA and 0.1% sodium azide in PBS for 30 min. Thecells were washed twice with PBS and analyzed by flow cytometry.

F-Actin Quantification. The amount of F-actin in activated CD4+ T cellsin the presence and absence of Salp15 was quantified byultracentrifugation and Western blotting. CD4⁺ T cells were activated asdescribed above for 2 min. Cells were lysed in HEPES buffer containingTriton X-100 (10 mM NaCl, 135 mM KCl, 2 mM MgCl₂, 2 mM EGTA, 10 mM HEPESpH 7.15, 1% Triton X-100, 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin, 1mM PMSF). The cell lysates were centrifuged for 5 min at 12,000×g at 4°C. in a table top centrifuge. The supernatant was then centrifuged in aBeckman ultracentrifuge, rotor TLA.100.2, for 30 min at 320,000×g at 4°C. The remaining pellet containing F-actin was solubilized in SDSloading buffer overnight on ice. The samples were subjected to SDS-PAGEfollowed by Western blotting. Equal amounts of total protein were loadedon the SDS-gel based on the total actin determined from a sample of thecell lysate.

Cell activation from DO11.10 transgenic and CD4-deficient mice. T-cellsfrom CD4-deficient and B6 mice (The Jackson Laboratory, Bar Harbor, Me.,U.S.A.) were purified by negative selection and activated for 72 hrsusing 5:g/ml of plate bound anti-CD3 and 1 μg/ml of anti-CD28 in thepresence or absence of 50:g/mL of Salp15. DO11.10 transgenic mouse (TheJackson Laboratory) CD4⁺ T cells were purified by negative selection asbefore and incubated in the presence of 106/mL of syngeneic mitomycinC-treated (50 μg/ml for 40 min at 37° C.) antigen presenting cells(APCs) with 10 μg/mL of ovalbumin in the presence of 0.5, 5.0 and 50μg/mL of Salp15 for 72 hrs. The supernatants were then collected andIL-2 measured by capture ELISA following the manufacturer's instruction(BD Biosciences Pharmingen).

Microtiter Binding assay. Purified sCD4 was coated overnight at 4° C. atthe indicated concentrations in 0.1 M sodium carbonate buffer (pH 9.5)in 96 well plates. Equimolar amounts of lysozyme were used as anon-specific control. The wells were washed, blocked with 10% FCS/PBSfor 1 hr at R.T., and incubated with Salp15 (0.4 μM) for 2 hr at 37° C.Salp15 binding was detected by incubation with horseradish peroxidase(HRP)-conjugated anti-His antibody for 1 hr followed by microwellperoxidase substrate (KPL). The reaction was stopped by adding TMB stopsolution (KPL). Chemically synthesized overlapping peptides of Salp15were purchased from GenScript Corporation (Piscataway, N.J., U.S.A). Tostudy the binding of Salp15 and peptides to sCD4, Salp15 (5 μg) anddifferent peptides (0.5 μg) were coated. The wells were washed andblocked prior to incubation with sCD4-HRP (Research Diagnostics Inc.,Flanders, N.J., U.S.A.). For competition assays, indicatedconcentrations of Salp15 or P11 were added to sCD4-HRP and incubatedwith plate bound P11 (0.1 μg). To estimate the effect of dithiothreitol(DTT) treatment of Salp15 on binding to sCD4, 0.5 μM Salp15 was treatedwith 10 mM DTT overnight and immobilized onto microtiter plate followedby blocking and incubation with sCD4-HRP.

Analytical gel filtration. Purified sCD4 (D1-D4, 10 μg) and Salp15 (20μg) were eluted through two SUPERDEX-200™ HR 10/30 gel filtrationcolumns (Amersham Pharmacia Biotech, Piscataway, N.J., U.S.A.) connectedin tandem using PBS as equilibration and elution buffer at a flow rateof 0.3 mL/min using an FPLC system (Amersham Pharmacia Biotech) equippedwith a liquid chromatography controller LCC-501 Plus and regulated usingFPLC director V1.10. To get the elution profile of the Salp15-CD4complex, Salp15 was incubated with sCD4 before passing it through thegel filtration column. Blue dextran was used to estimate the void volumeof the columns, while BSA, ovalbumin and lysozyme were used as molecularweight standards with elution volumes of 14.78 mL, 26.45 mL, 28.45 mLand 39.28 mL, respectively.

Native Gel electrophoresis. Purified sCD4 (D1-D4, 10 μg), Salp15 (10 μg)and sCD4 (10 μg)+Salp15 (10 μg) were diluted into 50% glycerol, 40 mMHEPES (pH 6.5) and 50 mM imidazole (pH 6.5). The native gel [5%Acrylamide:Bis (29:1), 40 mM HEPES (pH 6.5), 50 mM imidazole (pH 6.5)and 10% glycerol] electrophoresis was performed using a 40 mM HEPES (pH6.5) running buffer and 50 mM imidazole (pH 6.5) at 8 mAmps for 1.5hours at 4° C. The gel was stained with coomassie blue or transferred toIMMOBILON P® (Millipore) blotting paper for immunoblotting withanti-Poly His-HRP and anti-CD4.

Ellman assay. The solutions used for Ellman assay were solution A (37 mMsodium phosphate buffer, pH 8.0), solution B (7.4 mM sodium phosphatebuffer, pH 8.1), solution C (25 mM EDTA in solution B), solution D (10mM DTNB (Sigma) in solution A) and solution E (protein samples insolution B). The solutions to be measured on a spectrophotometer wereprepared as follows: (1) the reagent blank contained 240 μL C, 60 μL Dand 900 μL B; (2) the reaction mixture contained 20 μL C, 5 μL D and 75μL E; (3) the protein blank was prepared by mixing 20 μL A, 5 μL D and75 μL E; and (4) the buffer blank was made by mixing 240 μL A, 60 μL Dand 900 μL B. These solutions were incubated at 25° C. for 45 min beforereading the absorbance at 412 nm. The final absorbance, A was definedas:A=ADTNB−Aprotein  [Eq. 1]where ADTNB is the absorbance for solution 2 minus solution 1, andAprotein is absorbance for solution 3 minus solution 4. Theconcentration of free thiol in solution 2 was then calculated using theformula:A=εL[Salp15−SH]  [Eq. 2]where ε is equal to 14150 M−1 cm−1. The concentration of free thiol insolution 2 was corrected for dilution and percentage of free thiolcontent of Salp15 was calculated as:Free thiol %=[Salp15−SH]×100/[Salp15]  [Eq. 3]where the concentration of Salp15 was obtained using the Bradford assay.

Statistical analysis. Dissociation constants were determined bynon-linear curve fit. The results are expressed as the mean±S. E. of 3to 5 individual experiments.

Example 1 Salp15 Inhibits Early Protein Tyrosine Phosphorylation DuringT Cell Activation

During TCR signaling, PLCγ1 hydrolyzes phosphatidyl-inositol (4,5)-bisphosphate to inositol (1,4,5)-trisphosphate and diacylglycerol,leading to calcium mobilization and protein kinase-C activation,respectively (Rhee & Bae, 1997). It was first questioned whether PLCγ1activation, regulated by its phosphorylation at Tyr783 (Mustelin et al.,1990), was affected by Salp15 treatment of T lymphocytes.Phosphorylation of PLCγ1 was reduced in stimulated mouse CD4⁺ Tlymphocytes which were pretreated with Salp15, compared to controltreated cells (46%, FIG. 1A). This inhibition was also evident in theJurkat clone E6-1, a human T cell leukemia cell line, suggesting thatSalp15 is equally effective in causing immunosuppression in human Tcells.

To investigate whether the upstream signaling pathways resulting inPLCγ1 activation were also affected by Salp15 treatment, the tyrosinephosphorylation of membrane bound and cytosolic proteins induced by TCRligation in the presence or absence of Salp15 was examined. TCRligation-induced tyrosine phosphorylation of several proteins waslowered to different degrees by pretreatment of CD4⁺ T cells with Salp15(FIG. 1B and FIG. 7A). The molecular weights of these proteinscorresponded to those of Lck, Vav1, Lat, and Zap70, which was furtherconfirmed by assessing the phosphorylation status of some of thesesignaling components individually by using phospho-specific antibodies.The phosphorylation levels of Lat at Tyr¹⁹¹ (FIG. 1B), Lck at Tyr³⁹⁴ andZap70 at Tyr³¹⁹ (FIG. 1C) were reduced by Salp15 pretreatment. Salp15also inhibited TCR ligation-induced tyrosine phosphorylation of Vav1 asexamined by immunoprecipitation of total Vav1 followed by anti-pTyrimmunoblotting (FIG. 1C).

The reduction in tyrosine phosphorylation of Lck, Zap70 and Vav1 bySalp15 pretreatment was estimated to be about 56%, 67% and 58%,respectively by quantification of the specific bands. The inhibitoryeffects of Salp15 on TCR ligation-induced tyrosine phosphorylation werealso observed in Jurkat cells (FIG. 7B). These results suggest thatSalp15 exerts a specific decrease in tyrosine phosphorylation ofsignaling proteins upon activation of T cells by anti-CD3/CD28.

Example 2 Reduced Lipid Raft Re-Organization in Salp15-Treated ActivatedT Lymphocytes

Lipid rafts, which are discrete plasma membrane lipid microdomains richin glycosphingolipids, cholesterol and glycosphosphatidylinositol-linked proteins (Simons & Ikonen, 1997), play a central role inTCR-induced signal transduction by segregating signaling molecules inresting cells and providing a compartment for their association andactivation upon receptor engagement (Alonso & Millan, 2001). Otherwisedispersed over the cell surface as small domains, the lipid raftscoalesce in response to TCR engagement, amplifying and sustainingTCR-induced signals (Rodgers & Rose, 1996; Viola et al., 1999). Thus,whether Salp15 pretreatment affected lipid raft reorganization duringCD4+ T cell activation in response to anti-CD3/CD28 signals wasinvestigated. The redistribution of rafts was assessed usingAlexa-fluor⁵⁹⁴-labeled cholera-toxin B (CTB⁵⁹⁴) which binds to theganglioside GM1 enriched in lipid rafts. CTB⁵⁹⁴ staining of thestimulated CD4⁺ T cell membrane was confined in a dense cap structure,as described (Janes et al., 2000) in contrast to unstimulated T cells(FIG. 2A), indicating that rafts had aggregated during the activationprocess. Lipid raft clustering, however, was reduced by Salp15 treatmentprior to the stimulation with anti-CD3/CD28 in both murine cells (FIG.2A) as well as Jurkat cells (FIG. 7C).

Lipid raft re-organization is not exclusive for CD4⁺ T cells and is alsoinvolved in signaling processes in other cell types such as CD8⁺ Tcells, B cells and Fc receptor bearing cells. To examine the influenceof Salp15 in these cell types, receptor capping in T, B and phagocyticcells in response to anti-CD3/CD28, anti-IgM and Fc engagement,respectively, using fluorescent-labeled cross linking antibodies wereanalyzed. Salp15 did not affect lipid raft reorganization in B cells andmacrophages, while clustering was reduced in CD4⁺ T cells (FIG. 2B).Lipid raft reorganization during CD8⁺ T cell activation in response toanti-CD3/CD28 signals was also unaffected by Salp15 pretreatment asdetermined by CTB⁵⁹⁴ staining (FIG. 2C). Without wishing to be bound bytheory, this suggests that Salp15 does not affect lipid raftreorganization per se, but affects the CD4⁺ T cell specific upstreampathways leading to lipid raft clustering upon activation. Again,without wishing to be bound by theory, this inhibition of lipid raftclustering could partly explain the immunosuppressive effect of Salp15on T lymphocytes by inhibiting the amplification of primary signalingevents induced by TCR ligation.

Example 3 Salp15 Inhibits Actin Polymerization in Activating TLymphocytes

Lipid raft redistribution is regulated by critical cytoskeletal changesincluding polymerization of globular (G)-actin into filamentous(F)-actin (Valensin et al., 2002) that occur upon TCR/CD3 complexengagement. To determine whether Salp15 pretreatment affected actinpolymerization induced by T cell activation, Alexa-fluor₄₈₈-labeledphalloidin was used to visualize the extent of F-actin formation byconfocal microscopy. Phalloidin₄₈₈ staining was high in activated CD4⁺ Tcells compared to naïve cells (FIG. 3A), while the extent ofphalloidin₄₈₈ staining was reduced in activated murine CD4⁺ T (FIG. 3A)and Jurkat cells (FIG. 7D) pretreated with Salp15. This effect wasfurther investigated by examining F-actin levels in naïve and activatedCD4⁺ T and Jurkat cells. The amount of F-actin was reduced uponpretreatment of T cells with Salp15 (FIG. 3B and FIG. 7E). Flowcytometry analysis of F-actin formation in activated and naïve T cellsusing phalloidin₄₈₈ also revealed reduced F-actin content in cellspretreated with Salp15 (FIG. 3C). This effect was seen as early as 1minute of activation further confirming that the primary signalingevents upstream of lipid raft clustering were also affected by Sapl15.

Example 4 CD4 Acts as the Specific Receptor for Salp15 on T Lymphocytes

The inhibition of TCR-induced early signaling by Salp15 led to theinvestigation of the interaction of Salp15 with the key receptorsinvolved in T cell signaling using coimmunoprecipitation methodologies.Salp15 co-immunoprecipitated the CD4 co-receptor (FIG. 4A) but not otherkey components of the T cell receptor complex, particularly TCRβ, CD3εand CD28 from a Jurkat cell lysate (FIG. 8A). Further, Salp15 was foundto bind specifically to CD4⁺ T cells, while no staining of CD8⁺ cellswas detected by confocal microscopy (FIG. 8B). The simultaneousvisualization of CD4 staining and Salp15 binding on unstimulated andactivated CD4⁺ T cells by confocal microscopy revealed thecolocalization of both proteins on the plasma membrane (FIG. 4B).

To further confirm this direct association, a non-lymphocyte cell lineexpressing CD4 was used. Flow cytometry analysis showed an increase inSalp15 binding to HeLa-CD4 cells compared to non-transfected controls(FIG. 4C). The analysis by confocal microscopy of Salp15 binding on HeLaand HeLa-CD4 cells revealed specific Salp15 binding on HeLa-CD4, but notin untransfected cells (FIG. 8C). Furthermore, CD4 staining colocalizedwith bound Salp15 on HeLa-CD4 cells (FIG. 4D). Co-immunoprecipitationexperiments further confirmed the direct interaction of Salp15 with CD4(FIG. 4E). Immunoprecipitation of a HeLa-CD4 cell lysate with Salp15 ora control His-tagged-thioredoxin-fused tick saliva protein (Salp13, Daset al., 2001) using an anti-His Ab showed specific immunoprecipitationof CD4 (FIG. 8D). Binding of Salp15₄₈₈ to HeLa-CD4 cells was competed bypreincubating the cells with either unlabeled Salp15 or a polyclonalanti-CD4 antibody (FIG. 4F and FIG. 4G), substantiating the specificityof the interaction between Salp15 and CD4. The ability of two anti-CD4mAbs, MT310 (CD4/V1-specific, Sutor et al., 1992) and OKT4(CD4/D3-specific, Moore et al., 1992) to compete with Salp15₄₈₈ forbinding to HeLa-CD4 cells was also investigated. Preincubation ofHeLa-CD4 cells with mAb OKT4 failed to compete Salp15₄₈₈ binding, whileMT310 showed competition with Salp15 staining (FIG. 4G).

To further substantiate the role of CD4-Salp15 interaction in theimmunosuppressive effect of the tick saliva protein, splenic T cellsfrom CD4-deficient mice were stimulated (McCarrick et al., 1993) withanti-CD3/CD28 in the absence or presence of Salp15. Theimmunosuppressive activity of Salp15 was highly diminished in T cellsisolated from CD4-negative mice (33% inhibition) as compared to controlT cells (85% inhibition, FIG. 9A). Without wishing to be bound bytheory, these results indicate that the immunosuppressive action exertedby Salp15 is mediated through its specific interaction with the T cellco-receptor, CD4.

Example 5 The C-Terminal Peptide of Salp15 Associates with theOuter-Most Extracellular Domains of CD4

The binding of Salp15 with CD4 was next characterized. Salp15 showed asaturable binding to soluble (s) CD4 (containing the extracellular outertwo domains, D1-D2) in a microtiter assay compared to a non-specificcontrol (Lysozyme; FIG. 5A) with a dissociation constant (Kd) of 47 nM.Salp15 showed a saturable binding to a sCD4 preparation that containsall four extracellular domains (D1-D4) with a similar kinetics (FIG.10A) in correlation with the observed competition with the CD4/D1-domainspecific mAb MT310 (FIG. 4G).

To further verify the interaction between Salp15 and sCD4, the gelfiltration profile of a Salp15-CD4 solution was studied. TwoSuperdex-200 gel filtration columns in tandem were employed to firststudy the elution profile of Salp15. The elution profiles of activeSalp15 were markedly different from those of inactive preparations ofthe protein. Active Salp15 preparations consisted primarily of monomerfractions while inactive Salp15 forms contained predominantly dimer andtrimer populations (FIG. 11). The gel filtration profile of sCD4 (D1-D4)contained a single peak corresponding to monomeric sCD4 (FIG. 5B). Anadditional peak overlapping with the sCD4 peak was observed in aSalp15-CD4 solution compared to the profiles of Salp15 and sCD4 alone(FIG. 5B). The overlapping peaks were resolved into individual peaks byGaussian fit analysis, which showed the elution volume of the Salp15-CD4complex to be 26.38 mL compared to 27.53 mL for the sCD4 peak (FIG. 5B).The calculated molecular weight of the Salp15-sCD4 complex was 67±8 kDa,suggesting that the molar ratio of Salp15-CD4 interaction is 1:1. Thepeak height of Salp15-CD4 was directly related to monomer content ofSalp15 in different experiments. Another small peak seen at elutionvolume of 21.78 mL, which corresponded to an estimated molecular weightof 120 kDa (FIG. 5B) was not consistently observed.

The interaction of Salp15 with CD4 was also studied by native gelelectrophoresis. Both sCD4 (D1-D4) and Salp15 co-migrated when allowedto interact in solution followed by gel electrophoresis under nativeconditions and Western blot analysis (FIG. 5C). Salp15 alone could notbe visualized in the native gel together with sCD4 and sCD4-Salp15,probably due to a large difference in the mobility of Salp15 compared tothose of sCD4 and sCD4-Salp15.

In order to map the residues in the immunomodulatory protein thatinteract with sCD4, synthetic 20 amino acid-long peptides with 10 aminoacid overlaps spanning the entire Salp15 sequence (Table 1) weregenerated. Only the C-terminal peptide of Salp15 (P11, amino acids95-114) showed binding to sCD4 (FIG. 6A). The binding was saturable,with a K_(d) of 50 nM (FIG. 6B). P11 binding to sCD4 could be competedby adding increasing concentrations of the free peptide (1050=1.25 μM;FIG. 10B), suggesting a specific interaction. The binding of P11 to sCD4could also be competed by adding increasing concentrations of Salp15(FIG. 6C). These data demonstrated that Salp15 binds to CD4 through itsC-terminal residues.

In order to gain insight into the potential role of the cysteinespresent in Salp15 and P11 in their binding to CD4, two approaches werepursued. First, a shorter version of P11, P11-2 was used that contains 2cysteines (amino acids 103-114 of the Salp15 sequence). This peptidealso showed saturable binding with CD4 compared to a non-specificcontrol (PLP, amino acids 139-151; FIG. 10C). Second, a CD4-bindingmicrotiter assay was performed with plate-bound Salp15 that had beenpre-treated with 10 mM DTT, which resulted in equivalent binding levelscompared to CD4 binding to control Salp15 (FIG. 10D). The Ellmananalysis of Salp15 showed that the molecule contains around 11% of free—SH groups, suggesting the presence of disulfide bonds that may involvean average of 6 of the 7 cysteines present in the molecule. Withoutwishing to be bound by theory, together these results suggest that eventhough the protein seems to contain several disulfide bonds, binding toCD4 is not mediated by their presence in the terminal portion of themolecule.

P11 was then tested for any immunosuppressive effects on T cellactivation. The pretreatment of cells with P11 showed a dose dependentinhibition of CD4⁺ T cell IL-2 production (FIG. 6D), while P8 (whichshowed no binding to CD4; FIG. 6B) did not exert any inhibition of Tcell activation. To further confirm this effect, lipid raftreorganization in response to T cell activation was examined in cellspretreated either with P11 or P8. The pretreatment of cells with P11caused a reduction in lipid rafts clustering while P8 did not show anyeffect (FIG. 6E). Thus, the C-terminal region of Salp15 is involved inits binding to CD4 as well as exerting immunomodulatory effects in Tcells.

Discussion of Examples 1-5

CD4 plays a crucial role during T cell activation by facilitating theassociation of Lck with the CD3-TCR complex-associated ITAMS (Horejsi etal., 2004). The results disclosed in the EXAMPLES suggest that Salp15treatment prior to T cell stimulation impedes the proper activation ofthe Src kinase Lck, which is considered almost the first protein to getphosphorylated during TCR signaling (Horejsi et al., 2004). Lckphosphorylation results in the activation of downstream proteins by itstranslocation to lipid rafts since a palmitoylated form of Lck whichcompartmentalizes constitutively in lipid rafts has been shown to induceconstitutive activation of NF-AT and IL-2 production (Veri et al.,2001).

The results disclosed in the EXAMPLES have also confirmed that F-actinformation is reduced in activated T lymphocytes pretreated with Salp15.F-actin polymerization has been shown to be controlled by a number ofstimuli such as Vav1, through the activation of the small GTPases Rac1and cdc42 (Wulfing et al., 2000; Villalba et al., 2001). The guanineexchange factor activity of Vav1 is enhanced by its phosphorylation atseveral tyrosines through the Src family kinase Lck (Bustelo, 2000;Crespo et al., 1997; Riteau et al., 2003). The inhibition of TCRligation-induced protein tyrosine phosphorylation including those of Lckand Vav1, by Salp15 pretreatment explains the defective actinpolymerization during T cell activation and the improper lipid raftreorganization. Taken together, and without intending to be limited bytheory, these observations suggest that Salp15 affects T cell activationat the very beginning of TCR signaling, likely at the level of membranereceptors involved in TCR signaling. Indeed, the EXAMPLES provideresults showing that Salp15 interacts specifically with the CD4co-receptor, while no interaction was detected between Sapl15 and theTCR complex or the costimulatory molecule, CD28. Furthermore, Salp15interacts with the extracellular outer two domains of CD4 through itsC-terminal residues by a mechanism that is not likely to involve theseveral potential disulfide bonds found in the protein.

In contrast to co-engagement with the TCR, separate cross-linking of CD4either by antibodies specific for particular CD4 epitopes or by itsbinding to HIV gp120 has been shown to inhibit or markedly modify theoutcome of subsequent signals induced by TCR ligation (Bank & Chess,1985; Harding et al., 2002). Similarly, the selective ligation of CD4 invivo by specific antibodies in rodent models results in robustalloantigen-specific immunosuppression and induction of specifictransplantation tolerance (Benjamin & Waldmann, 1986; Laub et al.,2002). CD4⁺ T cell immunosuppression caused by Salp15 sharedsimilarities with these described interactions, such as the inhibitionof TCR induced tyrosine phosphorylation, calcium mobilization, NFAT andNF-κB activation that lead to IL-2 production.

However, Salp15 did not induce Lck phosphorylation in the absence of Tcell stimulation, in contrast to the described effect of gp120interacting with CD4 (Briand et al., 1997). Moreover, Salp15 does notaffect AP-1 DNA binding activation (Anguita et al., 2002) compared tothe ERK-mediated inhibition of AP-1 DNA binding activity observed incells treated with gp120 (Jabado et al., 1997). Indeed, Salp15 does notaffect AP-1 DNA binding activity T cells stimulated with lower doses ofanti-CD3 (2.5 μg/ml versus 5 μg/ml), even though the effect of theSalp15 is strongly dependent on the strength of the signal provided tothe activating T cells (Anguita et al., 2002), suggesting that thispathway is not affected by Salp15. While AP-1-mediated transcription isdependent on post-transcriptional modifications of its constituents,AP-1-mediated DNA binding activity does not require Tyr phosphorylationevents (Rincón et al., 1994). Meanwhile, the TCR-mediated signalsrequired for the binding activity of the complex remain unclear. PKCactivation is sufficient to trigger AP-1 DNA binding (Rincón et al.,1994) and ERK activation seems to play an important role in theactivation of the transcription factor through the expression of c-Fosand c-Jun (members of the AP-1 complex) (Chen & Davis, 2003; Cook etal., 1999). Unlike PLCγ1 activation, Salp15 does not impair ERKactivation in response to anti-CD3/CD28 stimulation (FIG. 9B) inagreement with a lack of effect of the immunosuppressor on AP-1 DNAbinding activity. Moreover, these data further discriminate the effectof Salp15 as compared to those of HIV gp120 and anti-CD4 cross-linking.

Since Salp15 interacts with the T cell co-receptor CD4 and inhibits theinitiation of TCR-mediated signals by blocking the activation of Lck, itseems likely that CD4 mediates, either fully or at least in part, theinhibitory effect of Salp15 on T cell activation. Moreover, the resultsset forth in the EXAMPLES suggest that in T cells, the majority of theeffect by Salp15 is mediated through its interaction with CD4. Comparedto the effect of Salp15 on IL-2 production in control B6 mice, theeffect in CD4-negative T cells was highly diminished. Furthermore,CD4-independent T cell responses such as INFγ-mediated phosphorylationof STAT1 were unaffected by Salp15 (FIG. 9C). The fact that the peptidecorresponding to the C-terminal amino acid residues of Salp15, whichshows strong binding to CD4, is capable of exerting the suppression of Tcell activation further supports that CD4 mediates the inhibitory effectof Salp15 on T cells. Salp15 inhibits the activation of CD4⁺ T cellsstimulated with anti-CD3/CD28, as well as through MHC II-CD4-dependentsignals (FIG. 9D and FIG. 6), suggesting that the effect of the proteinon T cell activation does not occur through interference with theinteraction between MHC II and CD4, but rather with the interaction andproper alignment of CD4 with the TCR complex, which is critical for Tcell activation. In this context, the following model is suggested, inview of the results disclosed in the EXAMPLES, for the immunosuppressiveaction of Salp15 on T cells. Upon contact with the membrane co-receptorCD4, Salp15 induces a conformational change in the receptor that blocksTCR-mediated Lck activation. This leads to the improper early activationof downstream signaling proteins resulting in defective F-actinpolymerization and a reduction in the clustering of signaling proteinsin lipid rafts, which are essential for the further amplification ofcell signals that result in IL-2 production and T cell proliferation.

I. scapularis ticks have been shown to modulate the host immune responsein many different ways, resulting in efficient transmission of severalclinically important pathogens (Wikel, 1999). These data are the firstknown example of an interaction between an arthropod protein and amammalian T cell co-receptor. This interaction clarifies the mechanismby which ticks withstand host immune responses during their encounterwith the host. This helps ticks maintain their presence on the host andthereby be more potent vectors of a number of pathogens. Since theeffect of Salp15 is not species specific, as it is evident in both mouseand human cells, Salp15 can be used in therapy of conditionscharacterized by CD4⁺ T cell responses, including autoimmune disordersand allogeneic transplant tolerance.

Introduction to Examples 6 and 7 Modulation of Relapsing-EAE by Salp15

MS is a chronic inflammatory disease of the central nervous system CNS.Self-reactive T cell activation and innate immune cell activation arethought to be major eliciting factors for the appearance of thesymptomatology associated with the damage of the myelin sheath. Evidenceexists that point to reactivation of memory T cells and/or epitopespreading (i.e., neoautoreactivity) to explain the chronicity and therelapsing-remitting clinical course often associated with the disease.

The study of MS has advanced dramatically with the use of differentrodent models, including rats and mice. The animals present withrelapsing episodes that are at least partially due to epitope spreading,which gives rise to the activation of new CD4⁺ T cells recognizing otherCNS peptides. Experiments carried out in the murine model have indicatedthat neoreactive CD4⁺ T cells are able to induce relapsing episodes.Therefore, the control of these relapsing episodes would provide a meansto evade progression of disease. Thus, inhibition of T cell activationin response to relapsing-associated epitopes during remission of theacute phase of the disease can prevent the appearance of relapsingepisodes.

Peptide specific disease therapy is difficult due to the diversity ofpeptides that can trigger relapsing episodes in humans. Alternativeapproaches include the inhibition of T cell activation using blockingantibodies to costimulatory molecules. As disclosed herein, Salp15polypeptides can be utilized as a treatment for patients with MS.

As disclosed herein, a protein from tick saliva (Salp15) inhibits CD4⁺ Tcell activation during encounter with the antigen, by preventing theproduction of the autocrine growth factor interleukin 2. Salp15 inhibitsTCR-mediated Ca²⁺ fluxes that lead to impaired activity of thetranscription factor NF-AT, without affecting the activation of effectorT cells. This protein can be used for the generation of immunotherapiesaimed at the prevention and/or treatment of relapsing MS.

In order to further validate the beneficial effects of Salp15 in theprevention of relapsing episodes of MS a murine model of MS (R-EAE) isused. The model is used to assess the effect of Salp15 administration onepitope spreading during experimental induction of murine EAE and assessthe effect of Salp15 on the clinical presentation of relapsing EAE.

Example 6 Effect of Salp15 Administration on Epitope Spreading DuringExperimental Induction of Murine EAE

Salp15 inhibits naïve CD4⁺ T cell activation and therefore, it canreduce or even prevent neoreactivity during the course of disease, suchas in MS for example. It has been demonstrated previously that epitopespreading has a role in relapsing episodes associated with R-EAE, ananimal model of human MS. Salp15 is tested to determine if it reduces oreven prevents the occurrence of activated CD4⁺ T cells in response tonew epitopes epitope spreading) during the course of experimental R-EAE.

salpl5 was amplified from a pBluescript vector using specific primers:5′-GAA AGC GGC CCA ACT AAA-3′ (SEQ ID NO: 18) and 5′-CTA ACA TCC GGG AATGTG-3′ (SEQ ID NO: 19). The PCR product was subcloned into thepMT/BiP/V5-His A vector (Invitrogen, Carlsbad, Calif., U.S.A.) andtransfected into Drosophila S2 cells (Invitrogen) in combination withthe hygromycin selection vector pCOHYGRO, for stable transfection. Thestable transformants were selected using 300 μg/ml hygromycin-B for 3-4weeks. The resistant cells were grown as large spinner cultures,switched to DES serum-free medium for 2 days and induced with coppersulfate to a final concentration of 500 μM for 4 days. The cells werethen centrifuged at 1,000×g for 5 minutes. The supernatant was used topurify the protein using the TALON™ metal affinity resin (Clontech, PaloAlto, Calif., U.S.A.). The protein was eluted using 100 mM imidazole,extensively dialyzed against PBS (pH 7.8) and concentrated bycentrifugal filtration through a 10 kDa filter (Millipore Corp.,Bedford, Mass., U.S.A.). The purity of the proteins was routinelychecked on SDS-PAGE and for activity on a CD4⁺ T cell activation assaywith anti-CD3/CD28 antibodies for 20-24 h.

SJL/J mice in groups of 18 are injected i.p. with 200 μg of PLP peptide139-151 (HSLGKWLGHPDKF; SEQ ID NO: 15) (Research Genetics, Huntsville,Ala., U.S.A.). Please note that the original Cysteine has beensubstituted by serine for stability purposes. This change does notinduce any alteration in the antigenicity of the peptide (Tuohy et al.,1989)) dissolved in an emulsion of 100 μL total volume of 60% CompleteFreund's Adjuvant and 40% sterile normal saline. One group of micereceives daily injections of 50 μg of recombinant Salp15 produced in aDrosophila expression system, starting day 10 after immunization. Thisregime permits an efficient in vivo activation of PLP₁₃₉₋₁₅₁-specificCD4⁺ T cell activation. Since Salp15 has no effect on effector CD4⁺ Tcells (Anguita et al., 2002.), its function is expected to becircumscribed to the activation of neoreactive CD4⁺ T cells. Anothergroup of mice receives daily injections of PBS. Controls include a groupof 3 animals that receive daily injections of Salpl5 starting the daybefore immunization with PLP₁₃₉₋₁₅₁. This group serves as a positivecontrol for Salp15 action and should prevent the activation ofPLP₁₃₉₋₁₅₁-specific CD4⁺ T cells in vivo. The experiments are repeatedat least twice to obtain statistically meaningful results.

At days 12 (including the 3 control mice treated with Salp15 over thecourse of the immunization), 14, 17, 20, 30 and 60 post-infection, 3mice from each group are sacrificed and analyzed as follows: splenicCD4⁺ T cells are purified by negative selection using biotinylatedantibodies against CD8a, Ly6G, Mac-I, B220, class II (I-A^(k) andI-E^(k)) and panNK molecules (BD Biosciences Pharmingen, San Jose,Calif., U.S.A.), followed by incubation with Avidin bound to magneticmicrobeads and passage through a magnetic column (Miltenyi Biotec,Auburn, Calif., U.S.A.). 10⁶ CD4⁺ T cells per ml are then incubated inthe presence of 10⁶ mitomycin C-treated (50 μg/ml, 37° C., 40 min)(Sigma Chemical Co., St. Louis, Mo., U.S.A.) syngeneic splenocytes withdifferent peptides (10 μg/ml) that have previously shown reactivity inthe murine model to assess the extent of epitope spreading during therelapse. These include: PLP₁₃₉₋₁₅₁ (immunization peptide), PLP₁₀₄₋₁₁₇,PLP₁₇₈₋₁₉₁ MBP₈₇₋₉₉ and MOG₉₂₋₁₀₆. These peptides include those thathave been associated with relapses in SJL mice (Vanderlugt et al.,2000). T cells are analyzed for 1) proliferation by [³H]-thymidineincorporation the last 18 hours of restimulation and 2) IFN(, and IL-4production at 40 h of restimulation, by ELISA. Antibodies specific formurine IFN(, and IL-4 (BD Pharmingen), are used, following themanufacturer's instructions with some modifications. Briefly, 96 wellELISA plates (ICN, Costa Mesa, Calif., U.S.A.) are coated with thecapture antibody (2 μg/ml) for 2 hours at 37° C. After blocking with PBSplus 10% FCS (PBS/FCS) overnight at 4° C., samples are applied andincubated 1 hour at 37° C. The biotinylated detection antibody (1 μg/ml)is added after washing the plates with PBS plus Tween 20 (0.5% v/v)(PBS/Tween). Quantitation of cytokine levels are made after incubatingthe plates with horseradish-conjugated avidin and adding the substratefor the enzyme (TMB, Kirkergaard and Perry Laboratories, Inc.,Gaithersburg, Md., U.S.A.). Plates are read at 450 nm after stopping thecolor developing reaction (TMB 1-Component Stop Solution, Kirkergaardand Perry Laboratories, Inc., Gaithersburg, Md., U.S.A.). The valuesindicated are calculated comparing the values obtained with thosederived using standard concentrations of recombinant mouse IFN(, andIL-4 (BD Pharmingen).

Peripheral responses to relapsing epitopes are assessed by the analysisof delayed type hypersensitivity (DTH). Four and seven days of theinitiation of the treatment with Salp15 or PBS in PLP₁₃₉₋₁₅₁-immunizedmice, groups of 5 animals are challenged in the left ear with 10 μg ofPLP₁₇₈₋₁₉₁, PLP₁₀₄₋₁₁₇, or MBP₈₇₋₉₉ and analyzed for swelling after 24and 48 hours. Average thickness is compared to the non-challenged earand control, non-immunized mice and assessed statistical significance bycomparison of the means with the Student T test.

The activity of Salp15 in vitro and in vivo suggests that the micetreated with the Salp15 protein will present lowered activation of CD4⁺T cells in response to new epitopes arising during the course ofexperimental disease. In contrast, reactivity of CD4⁺ T cells to theimmunizing peptide should remain intact, since Salp15 has no activity oneffector T cells (Anguita et al., 2002.).

A potential side effect of Salp15 in the mouse to contemplate is thepotential interference of Salp15 administration with selection processesof T cells in the thymus. In order to assess that the treatment with theprotein does not have undesirable side effects on the T cell repertoirethat would impair these mice to fight naturally occurring infections,thymic T cell populations are also analyzed. At the time of sacrifice,single cell suspensions of the thymi of the mice are obtained andanalyzed for 1) total cell numbers by counting total thymocytes bytrypan blue exclusion; 2) thymocyte subpopulations by FACS analysis ofCD4⁺ and CD8⁺ single positive (SP), double positive (DP) and doublenegative (DN: CD44⁺CD25⁻; CD44⁺CD25⁺; CD44⁻CD25⁺ and CD44⁻CD25)populations. These thymic populations represent different stages in Tcell development (from immature to mature: DNCD44⁺CD25⁻→CD44⁺CD25⁺→CD44⁻CD25⁺→CD44⁻CD25⁻→DP→SP) and can provide cluesas to whether Salpl5 interferes with the generation of appropriate Tcell repertoires; and 3) T (restimulation) and B cell (antibodyproduction) responses to a foreign antigen (Keyhole Lympet Hemocyanin,KLH) of mice after 2 weeks of treatment with Salp15. The mice aretreated for 2 weeks and immunized at the end of treatment with 10 μg ofKLH in complete Freund's adjuvant (CFA). The purpose of these controlsis to assure that the treatment does not impair the generation ofappropriate T cell repertoires that could compromise immune responsesto, for example, infectious agents.

Example 7 Effect of Salp15 on the Clinical Presentation of Relapsing EAE

Several lines of evidence point to a role of epitope spreading in MSprogression. The treatment of mice with experimentally induced EAE withagents that block CD4⁺ T cell activation (CTLA4-Ig, anti-B7 antibodies)or the induction of tolerance to peptides that are recognized during therelapsing episodes in mice, alter progression of the disease andrelapsing episodes (Vanderlugt et al., 2000; Karandikar et al., 2000;Miller et al., 1995). Some of these therapies are useful in the murinemodel, but difficult to apply in human disease because the exact natureof the antigens recognized during relapsing episodes is not known.Salp15 can reduce or even prevent epitope spreading during the course ofexperimental r-EAE and therefore, can reduce or even prevent progressionof disease. In order to further demonstrate Salp15 can ameliorateexperimental EAE, disease progression is analyzed in SLJ mice that aretreated with the immunosuppressive Salp15 protein.

SJL mice in groups of 10 are immunized with PLP₁₃₉₋₁₅₁, as before inEXAMPLE 6. The animals are monitored for the appearance and remission ofthe typical paralyzing symptoms. Mice are observed daily for clinicalsymptoms and scored as follows: 0, no evident disease; 1, loss of tailtone; 2, hind limb weakness; 3, paralysis of both hind limbs; 4,moribund state/death. Mice with intermediate clinical symptoms arescored in 0.5 increments. The results are tabulated for statisticalanalysis. All the observations are carried out at the same time of day(in the morning). If a mouse shows a grade of 4, the animal iseuthanized, as per IACUC recommendations. Clinical remission is definedas an improvement of at least one full clinical score that is sustainedfor at least two consecutive days.

Three days after the establishment of clinical remission, the mice aredivided in 2 groups. One group is treated with recombinant Salp15 everyday by i.p. injection (50 μg/mouse). The control group is treated withPBS. The animals are continuously monitored during 21 days for relapses.A clinical relapse is defined as an increase of at least one fullclinical score sustained for at least 2 consecutive days. The scores aretabulated and the groups compared for statistical significance.

The progression of disease is also evaluated by histological examinationof brains and spinal cords of immunized, treated and control mice.Groups of 3 mice are sacrificed after 7, 14 and 21 days of treatmentwith Salp15. Brains and spinal cords are fixed in formalin and embeddedin paraffin. Sections are then obtained and pretreated with 0.04% OsO₄and 1% H₂O₂ in 10% Triton X-100. The sections are then blocked withnormal goat serum and treated sequentially with a PLP monoclonalantibody (Harlan, Indianapolis, Ind., U.S.A.) and a peroxidase-labeledanti-mouse IgG antibody. The preparations will then be treated withdiaminobenzidine and 0.01% H₂O₂, 0.04% OsO₄ followed by washing withPBS. The sections are analyzed by microscopy.

Salp15 is a protein that can repress the activation of CD4⁺ T cells,including new epitopes that arise during experimental EAE. Epitopespreading has been associated with relapsing episodes in rodents andpotentially in humans. Salp15 is expected to inhibit the appearance ofrelapsing episodes in mice experimentally induced EAE. It is alsoexpected that a lower degree of demyelination as a result of treatmentwith Salp15 can be found, which would indicate that the treatment isable to prevent the progression of disease in the mice.

The use of Bordetella pertussis toxin for the induction of the diseasein mice, a common procedure for the induction of EAE in rodents ispurposely omitted. However, SJL mice develop R-EAE in the absence of thetoxin shortly after immunization with the eliciting peptide (Karandikaret al., 2000; Theien et al., 2001). Therefore, studies are conductedwithout the toxin. Nevertheless, studies in groups of mice treated withthis eliciting agent can be performed.

Example 8 Salp15 Competes with HIV gp120 for Binding to CD4

Ninety-six (96) well plates were coated with 0.05 uM sCD4 (D1-D2domains) in coating buffer (pH 9.6). The plates were washed 2× andblocked with 200 uL of PBS+10% FCS. Samples were then added containinggp120-HRP plus 0, 0.05, 0.5 and 5 uM Salp15 in PBS+10% FCS. KLH was usedas negative control at 5 uM. The plates were incubated for 2 hours atroom temperature and washed 4×, followed by incubation with horseradishperoxidase substrate and stop solution from KPL.

Results are shown in FIG. 12 and demonstrate a concentration dependentinhibition by Salp15 of gp120 binding to CD4. These results indicatethat Salp15 binds to a region in the CD4 molecule that overlaps with thebinging site for gp120.

Example 9 Salp15 Effect on gp120-CD4-Mediated Cell Fusion by LuciferaseAssay

106 HeLa-CD4 cells, clone 1022 (NIH AIDS Research & Reference ReagentProgram, Germantown, Md., U.S.A.) were plated in a 6-well plate andtransfected with the plasmid Blue 3′ LTR-luciferase (NIH AIDS Research &Reference Reagent Program) using the lipofectamin method (Invitrogen).

After 24 hours, the cells were trypsinized and mixed with HL2/3 cellsthat express HIV envelope proteins (NIH AIDS Research & ReferenceReagent Program), encoding Gag, Nef, Rev, Tat and Env proteins from HIV(0.2×106 HeLa-CD4+0.2×106 HL2/3) in a 24-well plate at a final volume of500 μL. The cells were incubated in the presence of 10 and 50 μg/ml ofSalp15. The positive control was anti-CD4 Ab and the negative controlwas anti-CD3.

The cells were harvested after 48 hours, washed with PBS and lysed with100 uL of Passive Lysis buffer (Promega) per well, for 15 min at RT. Thelysates were transferred to Eppendorf tubes, spun at 10,000×g andassessed for Luciferase activity and protein content by the Bradfordassay (BioRad). Luciferase activity is an indicator of cell fusionmediated by the interaction of HIV envelope proteins on HL2/3 cells andCD4 on HeLa-CD4 cells.

Results are shown in FIG. 13 demonstrating a concentration dependentreduction in cell fusion by Salp15. These data demonstrate Salp15interferes with fusion of cells expressing gp120 with cells expressingCD4 through interference of the Salp15 protein with gp120 binding toCD4.

REFERENCES

The publications and other materials listed below and/or set forth byauthor and date in the text above to illuminate the presently disclosedsubject matter, and in particular cases, to provide additional detailsrespecting the practice, are incorporated herein by reference. Materialsused herein include but are not limited to the following listedreferences.

-   Adelman et al. (1983). DNA 2:183.-   Alonso et al. (2004). J. Biol. Chem. 279:4922-4928.-   Alonso & Millan (2001). J. Cell. Sci. 114:3957-3965.-   Altschul et al. (1990). J Mol Biol 215:403-410.-   Andersson et al. (2000). Biopolymers 55:227-250.-   Anguita et al. (2002). Immunity 16:849-859.-   Bank & Chess (1985). J. Exp. Med. 162:1294-1303.-   Benjamin & Waldmann (1986). Nature 320:449-451.-   Berkow et al. (1997). The Merck Manual of Medical Information, Home    ed. Merck Research Laboratories, Whitehouse Station, N.J.-   Bodanszky (1993). Principles of Peptide Synthesis, 2nd rev. ed.    Springer-Verlag, Berlin/New York.-   Bolen & Brugge (1997). Annu. Rev. Immunol. 15:371-404.-   Briand et al. (1997). Virology 228:171-179.-   Burgdorfer et al. (1982). Science 216:1317-1319.-   Bustelo (2000). Mol. Cell. Biol. 20:1461-1477.-   Chen & Davis (2003). Mol. Cell. Endocrinol. 200:141-154.-   Chen et al. (1994). J. Clin. Microbiol. 32:589-595.-   Cook et al. (1999). Mol. Cell. Biol. 19:330-341.-   Corringer et al. (1993). J. Med. Chem. 36:166-172.-   Dalgleish et al. (1984). Nature 312:763.-   Crespo et al. (1997). Nature 385:169-172.-   Das et al. (2001). J. Infect. Dis. 184:1056-1064.-   Duch et al. (1998). Toxicol. Lett. 100-101:255-263.-   Ebadi (1998). CRC Desk Reference of Clinical Pharmacology. CRC    Press, Boca Raton, Fla.-   Ferreira & Silva (1998). Vet. Immunol. Immunopathol. 64:279-293.-   Fields & Noble (1990). Int J Pept Protein Res 35:161-214.-   Fleury et al. (1991). Cell 66(5):1037-1049.-   Freireich et al. (1966). Cancer Chemother. Rep. 50:219-244.-   Garbay-Jaureguiberry et al. (1992). Int J Pept Protein Res    39:523-527.-   Goodman et al. (1996). Goodman & Gilman's the Pharmacological Basis    of Therapeutics, 9th ed. McGraw-Hill Health Professions Division,    New York.-   Goverman & Brabb (1996). Lab. Anita. Sci. 46:482.-   Gribskov et al. (1986). Nuc Acids Res 14(1):327-334.-   Harding et al. (2002). J. Immunol. 169:230-238.-   Horejsi et al. (2004). Nat. Rev. Immunol. 4:603-616.-   Huang et al. (2000). Proc. Natl. Acad. Sci. USA 97:10923-10929.-   Janes et al. (2000). Semin. Immunol. 12:23-34.-   Karandikar et al. (2000). J. Neuroimmunol. 109:173.-   Katzung (2001). Basic & Clinical Pharmacology, 8th ed. Lange Medical    Books/McGraw-Hill Medical Pub. Division, New York.-   Kopecky & Kuthejlova (1998). Parasite Immunol. 20:69-74.-   Koretzky et al. (2003). Immunol. Res. 27:357-366.-   Krishnan et al. (2004). J. Immunol. 172:7821-7831.-   Kuchroo et al. (2002). Annu Rev Immuno. 120:101.-   Kyte et al. (1982). J Mol Biol 157:105.-   Laub et al. (2002). J. Immunol. 169:2947-2955.-   Lehmann et al. (1992). Nature 358:155.-   Lehrnann et al. (1993). Immunol. Today 14:203.-   Maddon et al. (1986). Cell 46:333-348.-   McCarrick et al. (1993). Transgenic Res. 2:183-190.-   McDougal et al. (1986). Science 231:382.-   McOmie (1973). Protective Groups in Organic Chemistry, Plenum Press,    London/New York.-   McRae et al. (1992). J Neuroimmuno 138:229.-   McRae et al. (1995). J Exp Med 182:75.-   Merrifield (1969). Adv. Enzymol. Relat. Areas Mol. Biol. 32:221-296.-   Michel et al. (1998). J. Biol. Chem. 273:31932-31938.-   Miller et al. (1995). Immunity 3: 739.-   Moore et al. (1992). J. Virol. 66:4784-4793.-   Motameni et al. (2004). Infect. Immun. 72:3638-3642.-   Mustelin et al. (1990). Science 247:1584-1587.-   Myung et al. (2000). Curr. Opin. Immunol. 12:256-266.-   Needleman et al. (1970). J Mol Biol 48:443.-   Pavone et al. (1993). Int J Pept Protein Res 41:15-20.-   PCT International Publication No. WO 93/25521.-   Qian & Weiss (1997). Curr. Opin. Cell Biol. 9:205-212.-   Ramamoorthi et al. (2005). Nature 436:573-577.-   Remington et al. (1975). Remington's Pharmaceutical Sciences, 15th    ed. Mack Pub. Co., Easton, Pa.-   Rhee & Bae (1997). J. Biol. Chem. 272:15045-15048.-   Ribeiro et al. (1995). Biochem. J. 308:243-249.-   Rincon & Flavell (1994). EMBO J. 13:4370-4381.-   Riteau et al. (2003). J. Exp. Med. 198:469-474.-   Rodgers & Rose (1996). J. Cell. Biol. 135:1515-1523.-   Sambrook et al. (2001). Molecular Cloning: A Laboratory Manual,    Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y.-   Schneider & Eberle (1993). Peptides, 1992: Proceedings of the    Twenty-Second European Peptide Symposium, Sep. 13-19, 1992,    Interlaken, Switzerland. Escom, Leiden.-   Schoeler et al. (1999). Exp. Parasitol. 92:239-248.-   Schröder & Lübke (1965). The Peptides. Academic Press, New York,    United States of America.-   Schwartz et al. (1979). Nuc Acids Res 6(2):745-755.-   Simons & Ikonen (1997). Nature 387:569-572.-   Smith et al. (1981). Adv Appl Math 2:482.-   Speight et al. (1997). Avery's Drug Treatment: A Guide to the    Properties, Choice, Therapeutic Use and Economic Value of Drugs in    Disease Management, 4th ed. Adis International,    Auckland/Philadelphia.-   Stewart & Young (1969). Solid Phase Peptide Synthesis, Freeman, San    Francisco.-   Sutor et al. (1992). J. Immunol. 149:1452-1461.-   Theien et al. (2001). J. Clin. Invest. 107:995.-   Thompson et al. (1994), Nucleic Acids Res 22(22):4673-4680.-   Tran et al. (2002). J. Immunol. 168:4293.-   Tung et al. (1992). Pept. Res. 5:115-118.-   Tuohy et al. (1989). J. Immunol. 142:1523.-   Urge et al. (1992). Carbohydr. Res. 235:83-93.-   Urioste et al. (1994). J. Exp. Med. 180:1077-1085.-   U.S. Pat. Nos. 4,244,946; 4,554,101; 5,234,933; 5,326,902;    5,578,629; 5,811,392; 5,811,512; 5,811,515; 5,817,757; 5,817,879;    5,834,228; 5,872,011; 6,015,561; 6,015,881; 6,031,071; 6,180,082.-   Valensin et al. (2002). Eur. J. Immunol. 32:435-446.-   Vanderlugt et al. (2000). J. Immunol. 164:670.-   Veri et al. (2001). Mol. Cell. Biol. 21:6939-6950.-   Villalba et al. (2001). J. Cell. Biol. 155:331-338.-   Viola et al. (1999). Science 283:680-682.-   Wethmur & Davidson (1968). J Mol Biol 31:349-370.-   Wikel (1999). Int. J. Parasitol. 29:851-859.-   Wikel & Bergman (1997). Parasitol. Today 13:383-389.-   Wulfing (2000). Proc. Natl. Acad. Sci. USA. 97:10150-10155.-   Yu et al. (1996). J. Exp. Med. 183:1777.-   Zhang & Samelson (2000). Semin. Immunol. 12:35-41.

It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. An isolated Salp15 polypeptide fragmentcomprising the amino acid sequence set forth in SEQ ID NO: 14, up to andincluding the amino acid sequence set forth in SEQ ID NO:
 13. 2. Theisolated Salp15 polypeptide fragment of claim 1, modified to be indetectably labeled form.
 3. A composition comprising a carrier and theisolated Salp15 polypeptide fragment of claim
 1. 4. The composition ofclaim 3, wherein the carrier is pharmaceutically acceptable for use inhumans.
 5. A fusion protein comprising the isolated Salp15 polypeptidefragment of claim 1, wherein the fusion protein comprises one or moreamino acids added to the N-terminus, the C-terminus, or both of theisolated Salp15 polypeptide fragment of claim 1.